Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation
Maksym Tsytlonok, Hugo Sanabria, Yuefeng Wang, Suren Felekyan, Katherina Hemmen, Aaron Phillips, Mi-Kyung Yun, Brett Waddell, Cheon-Gil Park, Sivaraja Vaithiyalingam, Luigi Iconaru, Stephen W. White, Peter Tompa, Claus A. M. Seidel, Richard Kriwacki
11 Dynamic anticipation by Cdk2/Cyclin A ‐ bound p27 mediates signal integration in cell cycle regulation Maksym
Tsytlonok *, Hugo
Sanabria *, Yuefeng
Wang *, Suren
Felekyan , Katherina
Hemmen , Mi ‐ Kyung
Yun , Brett
Waddell , Cheon ‐ Gil
Park , Sivaraja
Vaithiyalingam , Luigi
Iconaru , Stephen W. White , Peter
Tompa **,
Claus A. M. Seidel **, and Richard
Kriwacki ** VIB
Center for
Structural
Biology,
Vrije
Universiteit
Brussel,
Brussels,
Belgium. Department of Physics and
Astronomy,
Clemson
University,
Clemson,
SC,
USA. Lehrstuhl für
Molekulare
Physikalische
Chemie,
Heinrich ‐ Heine ‐ Universität,
Düsseldorf,
Germany. Department of Structural
Biology,
St.
Jude
Children’s
Research
Hospital,
Danny
Thomas
Place,
Memphis, TN USA. Current address:
Department of Radiation
Oncology,
West
Cancer
Center,
University of Tennessee
Health
Sciences
Center,
Memphis, TN USA Molecular
Interaction
Analysis
Shared
Resource,
St.
Jude
Children’s
Research
Hospital,
Danny
Thomas
Place,
Memphis, TN USA. Institute of Enzymology,
Research
Centre for
Natural
Sciences of the Hungarian
Academy of Sciences,
Budapest,
Hungary. Department of Microbiology,
Immunology and
Biochemistry,
University of Tennessee
Health
Sciences
Center,
Memphis, TN USA. *, ** these authors contributed equally to the work **Correspondence: [email protected] (PT), [email protected] (CAMS) and [email protected] (RK); Lead contact: [email protected] (RK).
Abstract p27
Kip1 (p27) is an intrinsically disordered protein (IDP) that folds upon binding to cyclin ‐ dependent kinase (Cdk)/cyclin complexes ( e.g., Cdk2/cyclin
A), inhibiting their catalytic activity and causing cell cycle arrest.
However, cell division progresses when stably
Cdk2/cyclin A ‐ bound p27 is phosphorylated on one or two structurally occluded tyrosine residues [tyrosines (Y88) and (Y74)] and a distal threonine residue [threonine (T187)]. These events trigger ubiquitination and degradation of p27, fully activating Cdk2/cyclin A to drive cell division. Using an integrated approach comprising structural, biochemical, biophysical and single ‐ molecule fluorescence methods, we show that Cdk2/cyclin A ‐ bound p27 samples lowly ‐ populated conformations that dynamically anticipate the sequential steps of this signaling cascade. “Dynamic anticipation” provides access to the non ‐ receptor tyrosine kinases, BCR ‐ ABL and
Src, which sequentially phosphorylate
Y88 and
Y74 and promote intra ‐ assembly phosphorylation (of p27) on distal T187.
Tyrosine phosphorylation also allosterically relieves p27 ‐ dependent inhibition of substrate binding to Cdk2/cyclin A, a phenomenon we term “cross ‐ complex allostery”. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression.
Intrinsic dynamics within multi ‐ component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease, as exemplified by hyper ‐ active BCR ‐ ABL and
Src in certain cancers. Keywords: intrinsically disordered proteins; signaling; intrinsic flexibility; cell cycle regulation; single ‐ molecule fluorescence; allostery; dynamic anticipation; protein NMR spectroscopy.
Introduction
Many eukaryotic proteins lack stable structures and exhibit conformational heterogeneity in isolation but fold into discrete conformations upon binding to other molecules (e.g. proteins, nucleic acids, small molecule ligands, metal ions, etc.) (Wright and Dyson,
These so ‐ called intrinsically disordered proteins (IDPs) often function to regulate complex biological processes (van der Lee et al., with their bound conformations exerting a basal level of regulatory control over their targets. However, this basal level of control can often be modulated, or switched, through post ‐ translational modifications to achieve complex regulatory behavior (Tompa, Van
Roey et al., as exemplified by the cell ‐ cycle regulator, p27 Kip1 (p27), which folds upon binding to cyclin ‐ dependent kinase (Cdk)/cyclin complexes that control cell division (Galea et al., The basal function of p27 is the inhibition of the kinase activity of Cdk/cyclin complexes, which can be relieved through phosphorylation of its tyrosine residue(s) by non ‐ receptor tyrosine kinases (NRTKs)(Chu et al., Grimmler et al., Relief of Cdk inhibition triggers a sequence of additional post ‐ translational modifications, including phosphorylation of T187 to activate a phosphodegron within the same p27 molecule, followed by its ubiquitination and degradation, triggering full activation of Cdk/cyclin complexes and progression to S phase during cell division. The existence of regulatory modification sites within bound and folded regions of p27 (and IDPs in general) raises a key question regarding how these sites become accessible for enzymatic modification in signal transduction. It has been previously suggested that regions of bound disordered proteins experience dynamic fluctuations (Grimmler et al., Tompa and
Fuxreiter,
Therefore, we hypothesized that regions within IDPs subject to function ‐ altering post ‐ translational modifications have evolved to dynamically sample different conformational states that provide accessibility to modifying enzymes. The ensuing enzymatic modifications remodel the conformational landscape of the IDP, enabling further changes and controlled integration of incoming signals. Herein, we tested this hypothesis by studying p27 bound to Cdk2/cyclin A by NMR spectroscopy, single ‐ molecule multiparameter fluorescence detection (smMFD) and other biophysical techniques, which revealed dynamic fluctuations (“dynamic structural anticipation”) that allow sequential phosphorylation of two of its tyrosines, Y88 (Grimmler et al., and Y88 and
Y74 (Chu et al., by the NRTKs,
Breakpoint cluster region ‐ Abelson murine leukemia viral oncogene homolog (BCR ‐ ABL) and
Src, respectively. We suggest that remodeling of dynamic structural ensembles of bound states by post ‐ translational modifications might be a general mechanism of signal integration by regulatory IDPs.
Results p27 phosphorylation restores
Cdk2 activity p27 is tethered to the Cdk2/cyclin A assembly via two discontinuous subdomains, D1 and D2, within its kinase inhibitory domain (KID); D1 binds to a hydrophobic surface patch on cyclin A and D2 binds to the active site of Cdk2 (for subdomain nomenclature, see
Figure for the structure of the assembly, see Figure
Phosphorylation of Y88 by BCR ‐ ABL was previously shown to partially relieve p27 ‐ dependent inhibition of Cdk2/cyclin A, triggering p27 ubiquitination and degradation, and full activation of Cdk2 to drive progression into S phase of the cell division cycle(Chu et al., Grimmler et al., Tyrosine located within D2 subdomain, is inserted into the ATP ‐ binding pocket of Cdk2 (Figure (Galea et al., Grimmler et al., Interestingly, p27 exhibits a second tyrosine within its KID,
Y74 (Figure and both
Y88 and
Y74 were previously shown to be phosphorylated by Src in a large fraction of hyper ‐ proliferative breast cancer cell lines (Chu et al., Dual phosphorylation of Y88 and
Y74 was shown to be associated with heightened Cdk2 activity (Chu et al., Grimmler et al., but the molecular mechanism of this effect was not investigated. In Cdk2 activity assays with p27 ‐ KID, in which Y88 (pY88 ‐ p27 ‐ KID) or both Y74 and
Y88 (pY74/pY88 ‐ p27 ‐ KID) are phosphorylated, we found that Y88 phosphorylation restored ~20 % of full Cdk2 activity at saturating concentrations of the inhibitor, as previously observed (Grimmler et al., whereas dual Y phosphorylation restored ~50 % of kinase activity (Figure Suppl.
Figure
Similar
Cdk2 reactivation was observed with
Y88 ‐ and Y74/Y88 ‐ phosphorylated full ‐ length p27 (Suppl. Figure
B).
Phosphorylation of tyrosine residues in p27 is sequential Next, we asked if these phosphorylation reactions occurred in a specific order, because both Y88 and
Y74 are buried against the surface of Cdk2 in the Cdk2/cyclin
A/p27 ‐ KID structure (Figure
PDB % and % solvent accessible surface area, respectively). We have previously shown that Y88 of p27 was accessible for phosphorylation by ABL kinase in cells and by ABL kinase domain (ABL ‐ KD) in vitro (when bound to Cdk2/cyclin A) (Grimmler et al., To address the issue of accessibility of Y74, we performed phosphorylation assays using Src kinase domain (Src ‐ KD) and pY88 ‐ p27 ‐ KID, as well as p27 ‐ KID constructs with
Y88 or both Y74 and
Y88 mutated to phenylalanine (Y88F ‐ p27 ‐ KID and
Y74F/Y88F ‐ p27 ‐ KID, respectively). In the absence of Cdk2/cyclin A, Y74 and
Y88 were phosphorylated equally by Src ‐ KD (Figure In the presence of Cdk2/cyclin A, Y74 was phosphorylated within p27 ‐ KID and pY88 ‐ p27 ‐ KID but not within
Y88F ‐ p27 ‐ KID (Figure
These results indicated that prior phosphorylation of Y88 is required for Y74 to become accessible for phosphorylation by Src ‐ KD, suggesting that the two tyrosine residues are sequentially phosphorylated.
How priming phosphorylation of Y88, with p27 tightly bound to Cdk2/cyclin A occurs, however, is enigmatic. Tyrosine phosphorylation of p27 exerts rheostat ‐ like control of Cdk2 activity and
T187 phosphorylation
Reactivation of Cdk2 through phosphorylation of Y88 stimulates
Cdk2 ‐ dependent phosphorylation of T187 within the flexible C ‐ terminus of p27 through a pseudo uni ‐ molecular mechanism within the Cdk2/cyclin
A/pY88 ‐ p27 ternary assembly (Das et al., Grimmler et al., Because
Src ‐ dependent dual Y phosphorylation of p27 is associated with reduced p27 protein levels in breast cancer cell lines (Chu et al., and because Cdk2 ‐ dependent phosphorylation of T187 is associated with p27 ubiquitination and degradation (Montagnoli et al., we next asked if dual Y phosphorylation of p27 within Cdk2/cyclin A complexes would further enhance intra ‐ assembly phosphorylation of T187. To this end, we compared the effect of mono Y and dual Y phosphorylation of p27 on T187 phosphorylation in vitro . As shown previously (Grimmler et al., a low level of T187 phosphorylation can be observed in Cdk2/cyclin
A/p27 assemblies (Figure
Suppl.
Figure this arises from bi ‐ molecular reactions (termed the “inter ‐ molecular” mechanism) between a small amount of free (and active) Cdk2/cyclin A and T187 within either free or Cdk2/cyclin A ‐ bound p27. T187 phosphorylation is enhanced by prior phosphate incorporation into pY88 ‐ p27 (Figure and dual Y phosphorylation causes a further ‐ fold increase of phosphate incorporation. The data exhibited the linear concentration dependence indicative of an intra ‐ complex mechanism (Grimmler et al., (Figure Thus, mono and dual Y phosphorylation of p27 exert rheostat ‐ like control of Cdk2 activity and phosphorylation of p27 on T187.
Reactivated
Cdk2: phosphorylation of intra ‐ assembly versus inter ‐ molecular substrates These observations show that Y phosphorylation of p27 tethered to Cdk2/cyclin A facilitates intra ‐ assembly phosphorylation of T187 (Figure whereas results with histone H1 showed facilitation of phosphorylation of a non ‐ specific substrate (Figure Next, we asked whether reactivation enhances phosphorylation of physiological substrates of Cdk2, such as retinoblastoma protein (Rb) and p107 (Adams, which are recognized by activated Cdk2/cyclin A via the same hydrophobic surface patch on cyclin A that binds the D1 subdomain of p27 KID.
Simultaneous monitoring of phosphorylation of T187 within p27 and of Rb or p107 showed that phosphorylation of Y residues within p27 enhanced Cdk2 ‐ dependent phosphorylation of both Rb and p107 (especially upon dual Y74/Y88 phosphorylation of p27), although to a lesser extent than that of the intra ‐ assembly substrate, T187 of p27 (Figure Suppl.
Figure
These results showed that activation of Cdk2 through Y phosphorylation of p27 primarily promoted intra ‐ assembly phosphorylation of T187 but non ‐ co ‐ assembled substrates also gain access to the active site of Cdk2 and/or the substrate binding pocket on cyclin A. Structural mechanism of Cdk2 reactivation by tyrosine phosphorylation of p27 The data discussed above suggest that p27 bound to Cdk2/cyclin A can sense and integrate activation signals from different NRTKs.
However, due to the tight binding of p27 to Cdk2/cyclin A, the structural mechanism that enables these phosphorylation events is unclear. We hypothesized that it could be linked with the distributed interaction interface between p27 ‐ KID and
Cdk2/cyclin A, which may enable phosphorylation to selectively interfere with small portions of this interface causing transient local (segmental) release of the bound inhibitor. In accord, NMR analysis previously showed that phosphorylation of Y88 displaces the C ‐ terminal half of the D2 subdomain containing Y88 from the
ATP binding pocket of the kinase (Grimmler et al., whereas dual Y74/Y88 phosphorylation displaces the entire D2 subdomain (Figure These results indicate that Y phosphorylation activates Cdk2 by making the active site sterically accessible. Next, we showed that the biochemical effect of this phosphorylation ‐ dependent displacement could be mimicked by deletion of residues ‐ from p27 ‐ KID (p27 ‐ KID ‐ C), which advanced our understanding of the extent (Suppl. Figure and structural basis (Suppl.
Figure
Suppl.
Table of partial Cdk2 activation.
Despite accessibility of the active site to ATP, only of full kinase activity was regained because p27 ‐ KID ‐ C altered the structure of Cdk2 near the active site. In particular, residues ‐ of p27 ‐ KID ‐ C formed an inter ‐ molecular ‐ strand with ‐ strand ( of Cdk2, displacing the strand that otherwise forms the G ‐ loop that binds the phosphates of ATP (Figure
Suppl.
Figure
This intermolecular ‐ strand can also be observed with p27 ‐ KID (Russo et al., where it reinforces full inhibition mediated by Y88. In the case of pY88 ‐ p27 ‐ KID and p27 ‐ KID ‐ C, in which the ATP binding pocket of Cdk2 is accessible, this feature explains the observed partial catalytic activity of the enzyme. Intrinsic dynamics in Cdk2 ‐ bound p27 enable tyrosine phosphorylation: insights from smMFD measurements To address the key question of how Y88, and subsequently
Y74, within the tight p27 ‐ Cdk2/cyclin A assembly becomes accessible for phosphorylation by Src ‐ KD, we introduced fluorescent dyes at several positions within p27, using native and non ‐ native cysteine residues ( cf. Figure and monitored their local and global structural and dynamic features using single ‐ molecule multiparameter fluorescence detection (smMFD) of freely diffusing protein molecules. To map local flexibility, single cysteine residues at positions and were labeled with BODIPY using a short linker (termed p27 ‐ C29, p27 ‐ C40, p27 ‐ C54, p27 ‐ C75, and p27 ‐ C93, respectively) and studied by single ‐ molecule fluorescence anisotropy (smFA) experiments. To additionally map global structural dynamics, three p27 constructs were prepared with pairs of Cys residues (at positions ‐ ‐ and ‐ and labelled with Alexa
Fluor and
Alexa
Fluor (termed p27 ‐ C29 ‐ p27 ‐ C54 ‐ and p27 ‐ C75 ‐ respectively) for use in single ‐ molecule Förster resonance energy transfer (smFRET) experiments.
The question of the enigmatic priming phosphorylation of Y88 was resolved by analyzing the local flexibility of BODIPY ‐ labeled pY88 ‐ p27 ‐ C93 by smFA experiments. The two ‐ dimensional histogram of fluorescence anisotropy r D vs. fluorescence ‐ weighted average lifetime ( τ D(0) f ) showed broad distribution of anisotropy values of single molecule bursts (Figure for all smFA fit parameters, see Suppl.
Tables ‐ We applied probability distribution analysis (PDA) to properly account for the shot noise in the histograms (Kalinin et al., PDA disclosed two underlying long lived (> ms) states – one of high ( r DH , light blue line, more rigid state) and another of low ( r DL , dark blue line, more flexible state) anisotropy. The single ‐ molecule data revealed an equilibrium, where, even in the absence of Y88 phosphorylation, this region samples a minor, flexible state (33 %), which increases in population (to %) significantly upon Y88 phosphorylation (Figure
The existence of this lowly populated, solvent ‐ exposed conformer of region ‐ of p27 ‐ KID provides an explanation for the accessibility of Y88 for phosphorylation by BCR ‐ ABL and
Src in cells and the corresponding kinase domains in vitro (Chu et al., Grimmler et al., smFA data for p27 labeled with BODIPY on positions C54 or C75 (Figure revealed further conformational features of D2 and also of the LH subdomain of p27 ‐ KID. In both regions, a major, highly populated state exhibited a high r D value ( r D > supporting the view that positions and are mostly rigid both before and after Y88 phosphorylation.
The minor state with a low anisotropy value ( r D ~ Figure
Suppl.
Figure increased in population upon dual Y74/Y88 phosphorylation of p27, markedly for position and slightly for position consistent with NMR results showing that the entire D2 subdomain was released upon dual Y phosphorylation (Figure Also in line with NMR observations are the smFA results for other regions of p27 KID as reported by C40 and
C29 within the D1 subdomain. C29, which can be resolved with NMR, exhibits a highly populated rigid (high r D ) state, whereas for position C40 (unresolved by NMR) a flexible (low r D ) state is the major population. For
C40, r D populations were nearly unchanged upon Y88 and
Y88 ‐ Y74 phosphorylation, whereas for
C29, there is some increase in the flexible state already upon Y88 phosphorylation (Figure
Suppl.
Figure To elucidate the structural context of the dynamic motions observed by smFA, we performed smFRET experiments and analyzed them by smMFD ‐ plots and correlation analysis (Figure Suppl.
Figure all determined FRET observables are compiled in Suppl.
Tables ‐ First, we displayed the data by plotting a two ‐ dimensional (2D) frequency smMFD ‐ histogram for the two FRET indicators averaged donor ‐ acceptor distance R DA E (derived from the intensity ratio of donor over acceptor signal) vs. the average donor fluorescence lifetime τ D(A) f , which will be shortened by FRET.
This representation allows us to directly assess the structural heterogeneity and dynamics of the sample on different time scales(Sisamakis et al., The smFRET data for p27 ‐ C54 ‐ bound to Cdk2/cyclin A (Figure showed a broadly distributed population peaking at R DA E values ~ Å and τ D(A) f ~ ns. The maximum population in the histogram is shifted to the right of the static FRET line (Figure green curve;
Suppl.
Table which is a hallmark for fast dynamic mixing of at least two different limiting states (Sisamakis et al., Moreover, the dynamic
FRET populations are very broad which indicates differently averaged populations in slow exchange. We applied PDA analysis to appropriately account for the broadening of the histograms due to shot noise and distinct acceptor brightness values (Kalinin et al., Kalinin et al., This way we could reveal at least two averaged conformational states: a highly populated, major state (83 %) with R DA E = Å (Figure dark blue), and a somewhat more extended minor state (17 %) with R DA E = Å (Figure light blue). We assign the major state to a p27 conformation, in which Y88 is bound within the ATP pocket of Cdk2 [as observed in the Cdk2/cyclin
A/p27 ‐ KID structure (Russo et al., and the minor state to a conformation in which Y88 was released from this pocket, giving a longer inter ‐ dye distance. This interpretation is confirmed by comparing the measured inter ‐ dye R DA E values with expected distances calculated from the X ‐ ray structure of p27 ‐ KID bound to Cdk2/cyclin A (1JSU) (Russo et al., (Suppl. Table which was extended by molecular dynamics simulations (Galea et al., for the unresolved (> residues of p27 (Figure To describe the dye behavior, we performed coarse grained accessible volume simulations (Suppl. Table (Sindbert et al., and compared the calculated interdye distances to the highly populated states of p27 ‐ C29 ‐ (and also of p27 ‐ C54 ‐ and p27 ‐ C75 ‐ see later). In all cases we found that the smMFD ‐ derived and simulated distances were in agreement (within %); hence, this state indeed represents p27 bound to Cdk2/cyclin A. We next observed that the minor state population increased to % upon Y88 phosphorylation.
Although ejected due to Y88 phosphorylation based on NMR data (Figure % of the molecules still exhibited the shorter R DA E values (Figure suggesting that the pY88 region transiently interacts with the ATP pocket.
Based on the smFA results, which showed no change after Y88 phosphorylation for position
C54 (Figure we conclude that the observed structural and local flexibility changes detected by smFRET with pY88 ‐ p27 ‐ C54 ‐ occur near Y88, rather than near position
Interestingly, despite a significant increase of the low ‐ anisotropy state of C54 upon
Y74 phosphorylation in smFA (Figure smMFD studies with the p27 ‐ C54 ‐ construct showed no major changes in the conformational features or populations of the two states following dual Y74/Y88 phosphorylation in comparison with the mono Y88 ‐ phosphorylated form (Figure Suppl.
Figure In addition, smFA data for BODIPY ‐ labeled pY74/pY88 ‐ p27 ‐ C75 indicated only a small change in the populations of the high and low anisotropy states in comparison with the corresponding mono Y phosphorylated construct (Figure In contrast, smFA indicated liberation of position C54 from its more rigid, bound conformation (Figure
Because
NMR results showed the release of the entire D2 subdomain from Cdk2 upon dual Y phosphorylation (Figure and smFRET results suggest that the region between C54 and
C93 maintains conformational topology similar to that of the bound state, we interpret these results by the existence of a partially populated secondary structure in this region (Sivakolundu et al., and/or its structural compaction in the free state, which restricts fluctuations of the residue pair, C54 and
C93, and also residue
Such compaction driven by hydrophobic residues within D2 subdomain has been observed by NMR (Iconaru et al., and is also supported by smFRET data with p27 ‐ C75 ‐ Here, the fraction of molecules with short R DA E increased strongly after mono pY88 phosphorylation. As smFA experiments conducted on p27 ‐ C75 did not show a change upon Y88 phosphorylation (Figure the observed change in distance distribution stems primarily from the liberation of the D2 subdomain, consistent with the NMR data (Figure
Direct observation of intrinsic p27 dynamics across broad timescales: dynamic anticipation Irrespective of the actual structural state of liberated D2 subdomain, the coexistence of two anisotropy ‐ and FRET ‐ populations and their position in the diagrams (Figure C; Suppl.
Figures ‐ already in the non ‐ phosphorylated complex indicates that p27 ‐ KID is in a slow dynamic exchange (on timescales identical to or slower than the diffusion time of ~ ms in the confocal volume, Suppl.
Table
S9) between a tightly Cdk2/cyclin A ‐ bound state ( cf. K D ‐ values in Table and a minor, loosely bound state. To study also the additional fast dynamic processes affecting p27 ‐ KID on its transition from the high FRET (HF) to the low FRET (LF) state and within each sub ‐ state, we computed the species ‐ autocorrelation (sACF) and ‐ cross correlation (sCCF) functions of the sub ‐ states depicted in Figure for the FRET pair p27 ‐ C54 ‐ C93 (SI
Section (Felekyan et al., The recovered dynamic structural fluctuations with relaxation times between ns and ms report on chain (60 ns) and local (~ µs, µs and µs, for details of the fFCS parameters, cf. Suppl.
Table dynamics. The fact that these dynamics are always present, even in the absence of phosphorylation, demonstrates the inherent dynamic anticipation of the complex, which is manifested in the liberation of local regions of p27 ‐ KID from the
Cdk2/cyclin A complex, making Y88 and
Y74 accessible for phosphorylation.
While for all
FRET ‐ pairs the relaxation times change only marginally upon mono or dual phosphorylation ( cf. Suppl.
Figure S6 and Table
S9), p27 ‐ KID liberation results in a marked increase in the relative fraction of the component showing fast chain dynamics in the sCCF and HF ‐ HF sACF and of slow dynamics in the LF ‐ LF sACF. Altogether, smFA and smFRET
PDA (Figures ‐ D) and species correlation functions (Figure consistently indicate a gradual shift of the binding mode from the “bound” state with a higher average FRET efficiency to a “more liberated” state with a lower average FRET efficiency due to progressive phosphorylation of Y88 and
Y74. In summary, based upon integration of biochemical, NMR and diverse smMFD data, p27 bound to Cdk2/cyclin A is best described as a highly dynamic assembly with multiple conformers in a multi ‐ level energy landscape, whose structural properties and binding interfaces are modulated by the degree of phosphorylation. Prior to phosphorylation, Y88 samples lowly populated solvent exposed conformations that enable its phosphorylation by NRTKs.
Once
Y88 is phosphorylated, the region ‐ is ejected from the ATP binding pocket of Cdk2 (Grimmler et al., which – as shown by smMFD data – can still transiently interact with the catalytic pocket of Cdk2.
This initial Y phosphorylation partially activates Cdk2 (Grimmler et al., and also exposes Y74 for phosphorylation, which causes displacement of subdomain D2 from Cdk2 and further kinase activation.
Interestingly, the displaced D2 subdomain appears to maintain conformational features similar to the Cdk2 ‐ bound state, consistent with past observations with isolated p27 ‐ KID (Iconaru et al., Sivakolundu et al., A critical feature of the Y phosphorylation ‐ dependent mechanism that modulates p27 ‐ dependent regulation of Cdk2 is that both pY88 ‐ and pY88/pY74 ‐ phosphorylated p27 remain tethered to the Cdk2/cyclin A assembly through binding of the D1 subdomain to cyclin A, enabling intra ‐ assembly phosphorylation of T187 within the flexible p27 C ‐ terminus. These results solve the enigma regarding how
Y88, which directly participates in Cdk2 inhibition, is made accessible for phosphorylation by NRTKs; this residue dynamically anticipates the Y phosphorylated state, enabling the critical first step of the multi ‐ step phosphorylation cascade that controls Cdk2 activity and cell division.
Thermodynamic and kinetic signatures of the tyrosine phosphorylation ‐ dependent p27 rheostat To add further dimensions to this model, we performed surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) experiments and monitored how Y phosphorylation affects interactions between p27 and Cdk2/cyclin A. Phosphorylation of Y residues did not affect the kinetics of p27 association with Cdk2/cyclin A but did slightly but progressively increase the rate of dissociation (Suppl. Figure Suppl.
Table similar results were obtained with p27 and p27 ‐ KID constructs.
These changes were associated with a phosphorylation ‐ dependent increase in apparent K D values but affinity for Cdk2/cyclin A and cyclin A alone remained high (apparent K D values of <2.9 nM and <52 nM, respectively), consistent with results from NMR and smMFD.
Next,
ITC was used to determine the number of residues of Y ‐ phosphorylated p27 that fold upon binding to Cdk2/cyclin A, as demonstrated previously for unmodified p27 (Lacy et al., The extent to which the folding of p27 upon binding is reduced is a surrogate for Y phosphorylation ‐ dependent displacement of p27 from Cdk2/cyclin A. For these experiments, we used constructs in which substitution of Y88 or both Y88 and
Y74 with glutamic acids mimicked the biochemical effects of phosphorylation. In agreement with the progressive partial release of p27 ‐ KID from
Cdk2/cyclin A upon phosphorylation, ITC shows different extents of folding upon binding to Cdk2/cyclin A for p27 and the mono and dual Y phosphomimetic forms, Y88E ‐ p27 and Y74E/Y88E ‐ p27 (Table Suppl.
Figure
With p27 and
Y88E ‐ p27, and residues folded, respectively, upon binding to Cdk2/cyclin A (values of , Table
This result suggests that a residue ‐ long region of subdomain D2 is displaced from Cdk2 in Y88E ‐ p27, consistent with the biochemical Cdk2 inhibition data, and past
SPR and
NMR data for p27 ‐ KID and pY88 ‐ p27 ‐ KID (Grimmler et al., on the extent of folding for p27 ‐ KID binding to Cdk2/cyclin A (Lacy et al., The K D values for these interactions were identical within error (4.9 ± nM and ± nM, respectively), which suggests a significant decrease in the entropic component and entropy ‐ enthalpy compensation for binding of Y88E ‐ p27 in comparison to p27 ( ‐ T S, Table
These observations provide strong support for increased dynamics in the bound state upon Y ‐ >E mutation. As demonstrated by smFRET experiments with the mono ‐ and dual Y phosphomimetic forms (Suppl. Figure they also show that C ‐ terminal residues outside of the N ‐ terminal KID experienced folding when the
KID (comprised of residues) folded upon binding to Cdk2/cyclin A. An even smaller number of residues folded when dual phosphorylation ‐ mimicking Y74E/Y88E ‐ p27 bound to Cdk2/cyclin A (48 residues; Table and the K D value increased to ± nM, consistent with the displacement of the entire D2 region from Cdk2 (Lacy et al., These conclusions were further supported by NMR data for the two phosphomimetic (Y ‐ >E) p27 ‐ KID mutants bound to Cdk2/cyclin A, which showed that the Y88 region and the D2 and LH subdomains, respectively, were displaced from Cdk2 by the Y74E and
Y74E/Y88E mutations (Suppl.
Figure
This displacement of the entire D2 subdomain from Cdk2 upon dual Y phosphorylation of p27 explains substantial reactivation of the kinase (50 % of full Cdk2 activity with pY74/pY88 ‐ p27 ‐ KID and % with Y74E/Y88E ‐ p27, Suppl.
Figure
Thus, the
SPR and
ITC data, with supporting biochemical and
NMR data, support the model discussed above in which Y phosphorylation displaces portions of inhibitory subdomain D2 of p27 from Cdk2, restoring partial kinase activity, while interactions between subdomain D1 and cyclin A are maintained. Tyrosine phosphorylation of p27 alters interactions with cyclin A through cross ‐ complex allostery Our collective results thus far reveal features of the conformational landscape of Cdk2/cyclin A ‐ bound p27 that involve interactions of its D2 subdomain with the kinase subunit of the assembly to regulate kinase activity. However, p27 also inhibits substrate phosphorylation by Cdk2/cyclin A through the binding of its D1 subdomain to a conserved surface on cyclin A, inhibiting substrate recruitment (Russo et al., Schulman et al., ( cf. Figure
Therefore, we considered the possibility that tyrosine phosphorylation within subdomain D2 could in some way alter interactions between subdomain D1 of p27 and cyclin A and thus affect this second inhibitory mechanism. To test this hypothesis, we utilized p27 constructs labeled on C29 alone (p27 ‐ or paired with C54 (p27 ‐ C29 ‐ in smMFD experiments. smFRET experiments showed a major (84%) state with a longer R DA E = Å and a minor (16%) state with a shorter R DA E = Å distance for p27 ‐ C29 ‐ bound to Cdk2/cyclin A (Figure Suppl.
Figure
After mono and dual phosphorylation, the fraction of the shorter R DA E state increased gradually, most probably due to partial release and compaction of the ‐ region. This is consistent with the dynamic release seen in smFA with BODIPY ‐ labeled p27 ‐ C29 bound to Cdk2/cyclin A (Figure Suppl.
Figure In all cases, the major state exhibited an r D value ≥ consistently with C29 binding to cyclin A, whereas the minor state has an r D value ≤ indicative of increased local mobility relative to the major state. Because
BODIPY ‐ labeled p27 ‐ C40 remained relatively constant upon mono and dual Y phosphorylation, the changes primarily occur at C29 and the fluctuations of subdomain D1 did not propagate into subdomain LH (Figure Suppl.
Figure We suggest that the increased population of the minor state in the smFRET data for the C29 ‐ dye pair with phosphorylation of both Y74 and
Y88 is due to allosteric effects that increase the dynamic fluctuations in the region around position Hence, we term this long ‐ range effect between D1 and D2 cross ‐ complex allostery. This long ‐ range effect may account, in part, for p27 Y phosphorylation ‐ dependent enhanced phosphorylation of the Cdk2 substrates, Rb and p107, and may be consistent with the non ‐ linear increase of their phosphorylation upon phosphate incorporation into p27 (Figure as opposed to that of the non ‐ specific substrate H1 (Figure Discussion Intrinsic dynamics within a multi ‐ component enzyme assembly is essential for the regulatory function of an IDP
Our study has addressed a key mechanistic question regarding the roles of IDPs as regulatory switches in signaling pathways; specifically, we addressed how sites within a bound IDP (p27 in complex with Cdk2/cyclin A) can become accessible to, and integrate, regulatory modifications despite apparent steric inaccessibility within a rigid structure (Russo et al., p27 experiences phosphorylation on two structurally inaccessible tyrosines, which signal p27 degradation and promote cell division (Chu et al., Grimmler et al., This is achieved by the intrinsic dynamics of p27, which, within the p27/Cdk2/cyclin A assembly is far from being static. Here, we have shown that the two events are sequential: the phosphorylation of Y88 is required for the subsequent phosphorylation of Y74, resulting in the D2 region to be fully displaced from Cdk2. By integrating biochemical (enzyme activity and substrate phosphorylation) and biophysical (NMR, X ‐ ray, smMFD, ITC, and
SPR) methods, we have discovered that the priming phosphorylation of Y88 is possible because this region structurally fluctuates between a major bound and a minor partially released state compatible with NRTK ‐ mediated phosphorylation (Figure Upon phosphorylation, the solvent ‐ exposed conformation of Y88 becomes more populated, and sterically enables phosphorylation of the minor, partially released state of Y74, which then displaces the entire D2 subdomain and facilitates intra ‐ complex phosphorylation of T187.
The activated phosphodegron mediates p27 poly ‐ ubiquitination and degradation and, ultimately, cell cycle progression. In all, tightly bound p27 within its complex with Cdk2/cyclin A dynamically anticipates the conformational changes caused by sequential Y phosphorylation (Figure Rheostat ‐ like control of a critical cell cycle kinase by an IDP
These structural fluctuations and remodeling of the conformational ensemble enable Cdk2/cyclin A ‐ bound p27 to actively integrate upstream NRTK signals with rheostat ‐ like precision. This rheostat has four settings: with unphosphorylated p27,
Cdk2 is “off”; with pY88 ‐ p27, Cdk2 is “20 % on”; and with pY74/pY88 ‐ p27, Cdk2 is “50 % on” (Figure to be turned “100 % on” by T187 phosphorylation and subsequent elimination of p27 by the ubiquitin ‐ proteasome system. The phosphorylation ‐ dependent functional properties of p27 are reminiscent of observations with the folded protein, dihydrofolate reductase (DHFR), that was shown using NMR spectroscopy to dynamically anticipate a sequence of structural changes that accompanied cofactor binding, substrate binding and product release (Boehr et al., Tuning structural fluctuations by post ‐ translational modification of p27 on Y88 and
Y74 through cross ‐ complex allostery may also trigger activation of cyclin A in the Cdk2/cyclin A complex, providing access to its substrate ‐ binding pocket to specific substrates (Figure This may be reflected in the non ‐ linear response of substrate modification to p27 phosphorylation, and may be essential for phosphorylation of external substrates, such as Rb and p107. Overall, our data are consistent with a model of sequential remodeling of the conformational energy landscape of bound p27 (Figure which might represent a general mechanistic theme of how IDPs sense, integrate and propagate signals within signaling pathways. Author contributions
M.T. prepared samples for smFL experiments, participated in smFL measurements and analyzed smFL data; H.S. performed smFL experiments, analyzed data and prepared figures;
Y.W. performed biochemical assays and
NMR experiments, and prepared associated figures, and prepared samples for
SPR experiments;
S.F. and
K.H. analyzed smFL data and prepared associated figures; M ‐ KY and S.W.W. performed X ‐ ray crystallography experiments and structure determination; B.W. performed and analyzed data from
SPR experiments and prepared associated tables and figures; C ‐ GP performed molecular biology and protein biochemistry experiments to prepare protein samples for smFL, SPR,
ITC and X ‐ ray diffraction experiments; S.V. performed analyzed data from
ITC experiments and prepared associated tables and figures;
L.I. performed
NMR experiments and prepared associated figures;
P.T. supervised
M.T. and performed smFL experimental design, analyzed data and wrote the paper;
C.A.M.S supervised
H.S. and performed smFL experimental design, analyzed data and wrote the paper; and
R.W.K. supervised the
St.
Jude co ‐ authors, conceived the project, performed experimental design, analyzed data and wrote the paper. Acknowledgements
This work was supported by the Odysseus grant
G.0029.12 from
Research
Foundation
Flanders (FWO) for
P.T.
M.T. was supported by a Marie ‐ Curie postdoctoral fellowship.
H.S. acknowledges a starting fund from Clemson
University.
C.A.M.S. was supported by the ERC
Advanced
Grant hybridFRET (671208).
R.W.K. was supported by US National
Institutes of Health (NIH) grants
R01CA082491 and
R01GM083159, a US National
Cancer
Institute
Cancer
Center
Support
Grant
P30CA21765 (at
St.
Jude
Children’s
Research
Hospital), and
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Legends
Figure Domain structure of p27. (A) Full ‐ length p27 contains the kinase inhibitory domain (KID), which is subdivided into domain (D1), linker helix (LH) and domain (D2). Tyrosine phosphorylation sites are labeled as Y74 and
Y88 and the threonine phosphorylation site is labeled as T187.
The labeling positions of cysteine residues are indicated as C29,
C40,
C54,
C75,
C93 and
C110. (B)
The structure of p27 in complex with Cdk2/cyclin A based on pdb:1JSU (Russo et al., and molecular dynamics simulations (Sivakolundu et al., Labeled sites are represented as circles, green only for smFA experiments and green/red circles where used in both smFA and smFRET experiments. Figure Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A and promotes phosphorylation of p27 on T187. (A) p27 ‐ KID completely inhibits the kinase activity of Cdk2/cyclin A toward histone H1 (squares; IC50 value, ± nM) while pY88 ‐ p27 ‐ KID (circles;
IC50 value, ± nM) and pY74/pY88 ‐ p27 ‐ KID (circles;
IC50 value, ± nM) are associated with and residual Cdk2 activity at saturating concentrations. (B) Y88 and
Y74 are sequentially phosphorylated by Src ‐ KD.
Various p27 ‐ KID constructs were used as sustrates for tyrosine phosporylation by Src ‐ KD in the absence (left lanes) or presence (right lanes) of Cdk2/cyclin A. The substrate, pY88 ‐ p27 ‐ KID, was prepared by prior treatment with ABL ‐ KD. (C)
Phosphorylation of Y88 (purple) and
Y74 and
Y88 (yellow) enables intra ‐ complex phosphorylation of p27 on T187 within ternary complexes with
Cdk2/cyclin A. T187 within unphosphorylated p27 is phosphorylated by Cdk2/cyclin A to a small extent due to intermolecular reactions (Grimmler et al., (D) The phosphorylation by Cdk2 of T187 is enhanced to a greater extent by phosphorylation of Y88 or Y74 and
Y88 within p27 (that is bound to Cdk2/cyclin A) than phosphorylation of the inter ‐ molecular Cdk2 substrates, Rb and p107. Fold enhancements of phosphorylation of T187 [in the presence of Rb (top) and p107 (bottom)], Rb and p107 are normalized to the extent of phosphorylation observed with unphosphorylated (on Y74 and
Y88) p27.
Figure Tyrosine phosphorylation ‐ dependent displacement of portions of the D2 region of p27 from Cdk2 mediates kinase reactivation. (A)
NMR analysis of the influence of tyrosine phosphorylation on interactions between p27 ‐ KID and
Cdk2/cyclin A. Chemical shift differences for residues in non ‐ phosphorylated (top) and tyrosine phosphorylated (pY88 ‐ p27 ‐ KID, middle; and pY74/pY88 ‐ p27 ‐ KID, bottom) p27 ‐ KID bound to Cdk2/cyclin A. Residues near
Y88 and within the entire D2 subdomain adopt free state ‐ like conformations in pY88 ‐ p27 ‐ KID and pY74/pY88 ‐ p27 ‐ KID, respectively. values were calculated using the equation: = [( H N ) + x ( N H ) ] . (B) Views of the region of Cdk2 (cyan) bound by p27 (orange) in various structures of Cdk2/cyclin A: Cdk2/cyclin
A/ATP (PDB left;
ATP is shown in ball and stick format), Cdk2/cyclin
A/p27 ‐ KID ‐ C (determined in this study, middle; a model of ATP is shown in semi ‐ transparent ball and stick format), and Cdk2/cyclin
A/p27 ‐ KID (PDB the position of ATP in the absence of p27 is shown in semi ‐ transparent ball and stick format). Both p27 ‐ KID ‐ C and p27 ‐ KID displace ‐ strand ( from the N ‐ terminal ‐ sheet of Cdk2 but, due to deletion of residues displaced by phosphorylation of Y88 in p27 ‐ KID ‐ C, the active site is accessible to ATP (modeled based upon the
Cdk2/cyclin
A/ATP structure).
However, displacement of the strand of Cdk2 (including residues of the G ‐ loop) in the Cdk2/cyclin
A/p27 ‐ KID ‐ C structure gives rise to an incompletely formed ATP binding pocket, consistent with the limited biochemical activity of Cdk2 within this complex.
Figure Local dynamic fluctuations of tyrosine residues within p27 bound to Cdk2/cyclin A. (A) Time ‐ window analysis ( Δ t =2 ms) for BODIPY ‐ labeled p27 ‐ C93 (blue) and pY88 ‐ p27 ‐ C93 (purple) bound to Cdk2/cyclin A is shown as a two ‐ dimensional histogram of scatter ‐ corrected fluorescence anisotropy ( r D ) vs. the fluorescence ‐ weighted average BODIPY fluorescence lifetime ( τ D(0) f ). One ‐ dimensional histograms are the projected distributions over a single variable. Probability distribution analysis (PDA) is used to fit the r D distributions with two shot ‐ noise limited states (high r D and low r D , light and dark colors, respectively). These values are used to calculate the proper rotational correlation times for each state using Perrin’s equation (Suppl.
Information, eq. (9);
Suppl.
Table ‐ Figure (B)
Global fits using
PDA for three time ‐ windows ( Δ t =1 ms, ms and ms) are presented for each single Cys variant (C29,
C40,
C54,
C75 and
C93).
The corresponding fractions and anisotropy values for the low and high r D are presented in light and dark colors, respectively. (C) Time ‐ window analysis ( Δ t =3 ms) for dual labeled p27 ‐ C54 ‐ (blue) and pY88 ‐ p27 ‐ C54 ‐ (purple) bound to Cdk2/cyclin A is shown as two ‐ dimensional histogram of the FRET averaged donor ‐ acceptor distance ( R DA E ) vs. the fluorescence ‐ weighted average fluorescence donor lifetime in presence of acceptor ( τ D(A) f ). One ‐ dimensional histograms are the projected distributions over a single variable. Grey shaded area indicates area of Donor ‐ only labeled molecules. PDA is used to fit R DA E distributions with two shot ‐ noise limited inter ‐ dye distances (low R DA E and high R DA E , light and dark colors, respectively). Horizontal lines are added for visual identification of both mean FRET states (Suppl.
Table ‐ Figure
Static
FRET lines calculated based on the fluorescence dye properties and PDA are shown in green (Suppl. Table No P: R DA E = (1/(3.8306/(0.0079* τ D(A) f +1.0179* τ D(A) f + ‐ ‐ (1/6) *53.0; pY88: R DA E = (1/(3.9997/(0.0202* τ D(A) f +0.9655* τ D(A) f + ‐ ‐ (1/6) *53.0. (D) Global fits using
PDA for three time ‐ windows ( Δ t =1 ms, ms and ms) are presented for each of the FRET ‐ samples (C29 ‐ C54 ‐ and C75 ‐ The corresponding fractions and inter ‐ dye distances for low R DA E and high R DA E are presented in light and dark colors, respectively. Error bars on R DA E represent the half ‐ width of the distribution of each state when fitting with PDA. (E)
The sACFs and sCCFs of filtered fluorescence correlation analysis for p27 ‐ C54 ‐ in complex with Cdk2/cyclin A without phosphorylation and with phosphorylated Y88 map the complex multi ‐ level dynamics of p27 reflecting the change of chain mobility (fit results see Suppl.
Table
Figure Structural dynamics within
Cdk2/cyclinA/p27 complex. p27 bound to Cdk2/cyclinA can be best described as a highly dynamic ensemble with multiple conformational states (approximated here by in a multi ‐ level energy landscape, whose structural properties and binding interfaces are tuned by the degree of phosphorylation. Prior to phosphorylation, Y88 already samples solvent exposed conformations that enable its phosphorylation by NRTKs (by
Abl).
This initial Y phosphorylation (of Y88, marked in purple) partially activates Cdk2, exposes
Y74 for phosphorylation (by
Src, marked in yellow), which causes displacement of subdomain D2 from Cdk2 (while preserving its local compact conformation), further kinase activation and intramolecular phosphorylation of T187, also facilitated by partial release of the substrate ‐ binding region on cyclin A. Flexibility is color coded from rigid (blue) to flexible (red). Table Equilibrium dissociation constant (K D ) values and thermodynamic parameters for the binding of p27, Y88E ‐ p27 and Y74E/Y88E ‐ p27 to Cdk2/cyclin A determined at °C using isothermal titration calorimetry (ITC). The ∆ Cp values were determined from H values measured at °C, °C, °C, °C, and °C and are reported in Suppl.
Figure p27 species binding to Cdk2/cyclin A K D (nM) ∆ G (kcal mol ‐ ) ∆ H (kcal mol ‐ ) ‐ T ∆ S (kcal mol ‐ ) ∆ Cp (cal mol ‐ K ‐ ) ( of residues that fold) p27 ± ‐ ± ‐ ± +39.2 ± ‐ ± Y88E ‐ p27 ± ‐ ± ‐ ± +26.9 ± ‐ ± Y74E/Y88E ‐ p27 ± ‐ ± ‐ ± +23.6 ± ‐ ± Supplemental
Information
Table of Contents
S1. Supplemental Methods ................................................................................................................ 4
S1.1.
Burst analysis and parametric lines for
FRET and anisotropy multidimensional histograms ............................................................................................................................................ 4
S1.2.
Probability
Distribution
Analysis (PDA) of single ‐ molecule Fluorescence
Anisotropy (smFA) and
Förster
Resonance
Energy
Transfer (smFRET) experiments . 6
S1.3.
Statistical uncertainties in PDA ........................................................................................... 7
S1.4.
Filtered
Fluorescence
Correlation
Spectroscopy .......................................................... 7
S1.5.
Accessible volume (AV) model and inter ‐ fluorophore distances ............................ 9 S2. Supplemental Tables ................................................................................................................. 10 Supplemental
Table Data collection and refinement statistics for determination of the structure of the Cdk2/cyclin
A/p27 ‐ KID ‐ C complex using X ‐ ray crystallography. ................................................................................................................................ 10 Supplemental
Table Fit parameters of smFA experiments obtained by PDA (Sections
S1.2,
S1.3). ...................................................................................................................... 11
Supplemental
Table Brightness correction for smFA experiments. .......................... 12
Supplemental
Table Rotational correlation time for smFA experiments. .............. 13
Supplemental
Table Fluorophore properties of dyes used in smFRET experiments and generated FRET lines according to S1.1 equations (3) and (7). .............................. 14
Supplemental
Table Fit parameters of PDA analysis for smFRET experiments. ... 16
Supplemental
Table F D /F A levels for High and
Low R DA E . ........................................... 17 Supplemental
Table Inter ‐ fluorophore distances of p27 FRET variants in complex with Cdk2/cyclin A based on accessible volume calculations (Section S1.5) and comparison with the “major” state in the non ‐ phosphorylated state. ........................ 18 Supplemental
Table Relaxation rate constants obtained by fFCS for Figure in main text (Section S1.4,
Equations (18) ‐ (20)). ....................................................................... 19 Supplemental
Table
Result of the analysis of triplicate measurements of p27 ‐ KID, pY88 ‐ p27 ‐ KID, and pY74/Y88 ‐ p27 ‐ KID binding to Cdk2/cyclin A, and separately to cyclin A or Cdk2, using surface plasmon resonance (SPR). .......................................... 21 S3. Supplemental Figures ............................................................................................................... 22
Supplemental
Figure Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A activity. ................................................... 22 Supplemental
Figure (next page). Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A and promotes phosphorylation of p27 on T187. .............................................................................................. 23
Supplemental
Figure Ejection of pY88 and the residue ‐ region from the Cdk2 active site is mimicked by truncation of p27 ‐ KID at residue .......................... 25 Supplemental
Figure Multidimensional smFA histograms of p27/Cdk2/cyclin A at various phosphorylation states. ................................................................................................. 26 Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/cyclin A at various phosphorylated states. ............................................................................................. 28 Supplemental
Figure Filtered
FCS
Species auto and cross ‐ correlation (sACF and sCCF) function of smFRET experiments for Cdk2/cyclin
A/p27 samples. .................... 30
Supplemental
Figure Representative results for varied concentrations of Cdk2/cyclin A, cyclin A, or Cdk2 binding to p27 ‐ KID, pY88 ‐ p27 ‐ KID, or pY74/Y88 ‐ p27 ‐ KID separately immobilized on the sensor surface. ................................................... 32 Supplemental
Figure (next page). Representative binding isotherms for injected p27 (A),
Y88E ‐ p27 (B), or Y74E/Y88E ‐ p27 (C) binding to Cdk2/cyclin A at temperatures from °C to °C recorded using isothermal titration calorimetry (ITC). ..................................................................................................................................................... 33 Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/Cyclin A with phosphomimetic variants. .................................................................................................. 34 Supplemental
Figure
The effects of mono and dual tyrosine phosphorylation on regulation of Cdk2 by p27 can be mimicked by mutation of Y88, and
Y74 and
Y88, to glutamate (E). .............................................................................................................................. 36 Online content
Methods, along with any additonal
Extended
Data display items and
Source
Data, are available in the online version of the paper. References unique to this section only appear in the online paper. Online
Methods
Protein expression and purification cDNA for residues (p27 ‐ KID), and (full length) of human p27 were sub ‐ cloned into pET28a (Novagen). A p27 construct in which Tyrosine was mutated to F (Y89F) was used in our studies because cellular ABL phosphorylates only
Y88 but the
ABL kinase domain (ABL ‐ KD) phosphorylates both
Y88 and
Y89 in vitro (Grimmler et al., Non ‐ physiological phosphorylation of Y89 is eliminated with the p27 ‐ KID ‐ Y89F construct, hereafter referred to as p27 ‐ KID. p27 ‐ KID ‐∆ C was prepared by deletion of residues ‐ from p27 ‐ KID in the pET28a expression vector. To mimic tyrosine phosphorylation, we prepared constructs with tyrosine (Y88) or both Y88 and tyrosine (Y74) mutated to glutamate (Y88E or Y88E/Y74E) using site directed mutagenesis. pET28a ‐ based expression vectors for p27 constructs with one or two non ‐ native Cys residues for smMFD experiments were prepared using site ‐ directed mutagenesis starting with p27 ‐ Y89F.
First, four wild ‐ type cysteine (Cys) residues were mutated to Ala or Ser, as follows: Cys to Ala,
Cys to Ala,
Cys to Ser, and
Cys to Ser (termed
Cys ‐ less p27). One construct was prepared with only the latter three mutations, leaving a single native Cys residue at position (p27 ‐ C29).
Then, constructs with one or two non ‐ native Cys residues were prepared in the Cys ‐ less p27 mutant background, as follows: Glu to Cys, p27 ‐ C40;
Glu to Cys, p27 ‐ C54;
Arg to Cys, p27 ‐ C93;
Cys and Glu to Cys, p27 ‐ C29 ‐ Glu to Cys and
Arg to Cys, p27 ‐ C54 ‐ and Glu to Cys and
Ser to Cys, p27 ‐ C75 ‐ The various mono ‐ and dual ‐ Cys mutant p27 constructs were shown to exhibit Cdk2/cyclin A inhibitory activity indistinguishable from that of p27 ‐ Y89F (data not shown).
The various p27 constructs, human
Cdk2,
T160 ‐ phosphorylated Cdk2, and truncated human cyclin A (residues of human cyclin A) were expressed in E. coli and purified as described (Lacy et al., The
SH3 and kinase domains of murine ABL (termed
ABL ‐ KD), and the C ‐ terminal domain of chicken Src (residues ‐ termed Src ‐ KD), were amplified by PCR, sub ‐ cloned into pET28a, expressed, and purified from E. coli using established procedures (Seeliger et al., The C ‐ terminus of human Rb (residues ‐ was sub ‐ cloned into pET28a with an N ‐ terminal, non ‐ cleavable fusion tag (NusA), expressed and purified from E. coli using Ni ‐ affinity chromatography (termed Rb).
The C ‐ terminus of human p107 (residues ‐ was expressed with glutatione S ‐ transferase (GST) and (His) fusion tags in E. coli and purified with a GST affinity column (termed p107). p27 constructs phosphorylated on Y88 were prepared through incubation with
ABL ‐ KD at °C for hours followed by Fe ‐ affinity chromatography. p27 constructs doubly phosphorylated on Y74 and
Y88 were prepared through incubation with
ABL ‐ KD followed by incubation with Src ‐ KD.
Isotope ‐ labeled samples of p27 ‐ KID and p27 for
NMR experiments were prepared in E. coli as previously described (Grimmler et al., using MOPS ‐ based minimal media (Neidhardt et al., and N ‐ ammonium chloride, H/ C ‐ glucose, and H O. In Vitro
Cdk2 kinase activity assays and kinetic studies of T187 phosphorylation
The inhibitory profiles of p27 and p27 ‐ KID constructs in which either Y88 or Y74 and
Y88 were phosphorylated or mutated to glutamate were determined by measuring in vitro Cdk2/cyclin A kinase activity toward histone H1 while titrating the different p27 species (Grimmler et al., IC values were determined using nonlinear regression analysis using Graphpad
Prism software.
Kinetic analysis of the T187 phosphorylation reaction was performed as described previously (Grimmler et al., with five different concentrations (0.25, and µM) with ternary complex of p27, pY88 ‐ p27 or pY74/pY88 ‐ p27 with cyclin A/Cdk2.
These reagents were equilibrated with γ‐ [ P] ‐ ATP for four time intervals (20, and min) and were followed by analysis using SDS ‐ PAGE and phosphoimager analysis (GE
Healthcare,
Piscataway,
NJ) of PO ‐ T187 within the p27 species. To compare the influence of p27 tyrosine phosphorylation on Cdk2 ‐ dependent phosphorylation of the intra ‐ complex substrate, T187, and two inter ‐ molecular substrates, Rb and p107, we performed the T187 phosphorylation reactions (using and M of the ternary complexes) described above in the presence of equimolar amounts of either Rb or p107 for minutes. The phosphorylation on T187 and either Rb or p107 was quantitated by analysis using SDS ‐ PAGE and phosphoimager analysis. In addition, to determine the order of phosphorylation of Y74 and
Y88 of p27, we performed phosphorylation reactions using µM Src ‐ KD, µM p27 species (p27, Y88F ‐ p27 or pY88 ‐ p27), µM Cdk2/cyclin A and γ‐ [ P] ‐ ATP.
The phosphorylation of tyrosine residues of p27 was determined using SDS ‐ PAGE and phosphoimager analysis. X ‐ ray crystallography p27 ‐ KID ‐Δ C and the Cdk2/cyclin A binary complex were mixed at a mole ratio and concentrated to ~15 mg/ml in mM HEPES, pH mM DTT, and mM NaCl by ultrafiltration. The ternary
Cdk2/cyclin
A/p27 ‐ KID ‐Δ C complex was isolated using gel filtration chromatography (Superdex GE Healthcare,
Piscataway,
NJ) using the same buffer followed by concentration by ultrafiltration to mg/ml. Crystals of Cdk2/cyclin
A/p27 ‐ KID ‐Δ C were grown by the hanging drop vapor diffusion method using the following reservoir solution: M HEPES, pH M lithium sulfate, and mM DTT.
The hanging drops contained equal volumes of the reservoir and the protein solution (15 mg/ml in gel filtration buffer) and were equilibrated against the reservoir solution at °C. Crystals were cryoprotected with glycerol and flash frozen in liquid nitrogen. Diffraction data were collected at the Southeast
Regional
Collaborative
Access
Team (SER ‐ CAT) ‐ BM beamline at the Advanced
Photon
Source and processed using
HKL2000 (Otwinowski and
Minor,
The structure was determined by molecular replacement using MOLREP (Vagin and
Teplyakov, with the
Cdk2/cyclin
A/p27 ‐ KID structure (PDB ID as a search model. The structure was refined and optimized using
REFMAC (Murshudov et al., PHENIX (Adams et al., and COOT (Emsley and
Cowtan,
Data collection and refinement statistics are summarized in Suppl.
Table The atomic coordinates for the
Cdk2/cyclin
A/p27 ‐ KID ‐Δ C complex have been deposited and validated by the Protein
Data
Bank (PDB for release upon publication.
NMR spectroscopy H/ N ‐ labeled p27 ‐ KID constructs (p27 ‐ KID, pY88 ‐ p27 ‐ KID and pY74/pY88 ‐ p27) and their complexes with Cdk2/cyclin A were analyzed using NMR spectroscopy at MHz using methods described previously (Grimmler et al, Briefly, the samples were dissolved in mM potassium phosphate, pH mM arginine, (v/v) H O, mM DTT ‐ D and (w/v) sodium azide. The isolated p27 ‐ KID constructs were analyzed using H ‐ N HSQC and their complexes with
Cdk2/cyclin A using H ‐ N TROSY at °C using a Bruker
Avance
MHz spectrometer equipped with cryogenically ‐ cooled TCI probe.
The spectra were interpreted based upon previously established resonance assignments (Grimmler et al., The
NMR experiments with the H/ N ‐ labeled Y88E and
Y74E/Y88E variants of p27 ‐ KID were performed similarly and resonances assigned by inspection. Assignment ambiguities were resolved through analysis of HNCA,
HN(CO)CA,
HNCACB,
HN(CO)CACB spectra of the isolated C/ N ‐ labeled Y to E mutant p27 ‐ KID constructs.
Surface plasmon resonance
Binding studies were performed at °C using a BIACORE optical biosensor (GE
Healthcare).
Unphosphorylated and tyrosine ‐ phosphorylated p27 ‐ KID constructs were covalently attached to a carboxymethyl dextran ‐ coated gold surface (CM ‐ Chip; GE Healthcare).
The carboxymethyl groups of dextran were activated with N ‐ ethyl ‐ N ´ ‐ (3 ‐ dimethylaminopropyl) carbodiimide (EDC) and N ‐ hydroxysuccinimide (NHS), and p27 ‐ KID constructs were attached at pH in mM sodium acetate. Any remaining reactive sites were blocked by reaction with ethanolamine. The kinetics of association and dissociation were monitored at a flow rate of μ l/min. Cdk2/cyclin A, cyclin A, and Cdk2 were prepared in mM Tris (pH mM NaCl, mM EGTA, mM DTT, mg/mL bovine serum albumin, and
Tween20.
Binding was measured for concentration ranges of pM – nM for Cdk2/cyclin A, – nM for cyclin A, and nM – µM for Cdk2. To account for injection artifacts, a series of sensorgrams was recorded throughout the experiment after injecting only buffer (blank injections). The chip surface was regenerated with a s injection of M guanidine ‐ HCl.
Data reported are the difference in SPR signal between the flow cells containing the p27 ‐ KID constructs and a reference cell lacking these constructs. Additional instrumental contributions to the signal were removed by subtraction of the average signal of the blank injections from the reference ‐ subtracted signal (Myszka, Triplicate injections were made at each concentration, and the data were analyzed globally by simultaneously fitting association and dissociation phases at all concentrations using the program Scrubber2 (Version
BioLogic
Software).
The kinetic rate constants were determined by fitting the data to a (Langmuir) interaction model. Equilibrium dissociation constants ( K D ) were calculated as the quotient k d / k a . Isothermal titration calorimetry
Thermodynamic parameters for the interaction of p27, Y88E ‐ p27 and Y74E/Y88E ‐ p27 with Cdk2/cyclin A were measured using a MicroCal auto ‐ iTC (Malvern Instruments) isothermal titration calorimeter.
Protein samples were exchanged into mM HEPES (pH mM NaCl and mM Tris (2 ‐ carboxyethyl) phosphine prior to the experiments. Titrations were performed by first injecting l of µM p27 or p27 Y to E variants into a solution of M Cdk2/cyclin A binary complex, followed by additional l or l injections. Experiments were carried out from C to C in intervals of C. Results were analyzed using
Origin software (OriginLab).
Equilibrium dissociation constant values (K D ) and thermodynamic parameters were determined fitting the data to a single ‐ site binding model using a nonlinear least ‐ squares fitting algorithm. The reported values are averages and standard deviations of the mean for three replicates. The values of the heat capacity change ( ∆ Cp) associated with the different p27 species binding to Cdk2/cyclin A were determined as the slope of the temperature dependence of the enthalpy change of binding ( ∆ H) values and the numbers of residues that folded upon binding ( ) were determined using the formalism of Spolar and
Record (Spolar and
Record, as previously described (Lacy et al., Production of dye ‐ labeled p27 for single ‐ molecule fluorescence studies We prepared p27 constructs in which most or all native cysteine (Cys) residues (at positions and were replaced by serine or alanine, and non ‐ native Cys residues were introduced to allow specific labeling with fluorescent dyes. Before labeling the proteins, all buffers were sterile filtered and degassed. p27 was concentrated to ‐ μ M in buffer A (20 mM Tris ‐ HCl pH mM NaCl) with mM DTT. ml of concentrated protein were loaded onto PD10 column and the protein was eluted with freshly degassed ml of buffer A without DTT.
The eluted p27 was first labeled with the acceptor
Alexa
Fluor maleimide fluorophore (Invitrogen) at a ratio, followed by labeling with the donor Alexa
Fluor maleimide fluorophore (Invitrogen) at ratio. The dual ‐ labeled p27 was separated from the homo ‐ labeled and unlabeled species using ion ‐ exchange chromatography. p27 Y88E and
Y88E/Y74E mutants were prepared in the same way. The presence of energy transfer was analyzed upon excitation at nm of the donor and acceptor dyes at nm and nm, respectively, using Perkin
Elmar
LS55
Luminescence
Spectrometer.
Single ‐ cysteine variants for anisotropy measurements were labeled with ‐ access of BODIPY ‐ FL or Alexa
Fluor (Invitrogen) and purified as above. Labeled p27 was then analyzed by gel ‐ filtration chromatography at µM protein concentration; the protein eluted at a volume expected for the monomer without any evidence of oligomerization or degradation. Labeled p27 was phosphorylated with
ABL ‐ KD and Src ‐ KD in the same way as non ‐ labelled protein. To ensure the maximal efficiency of phosphorylation, phosphorylated p27 was separated from non ‐ phosphorylated p27 using Pro ‐ Q® Diamond
Phosphoprotein
Enrichment
Kit (Invitrogen) and the results were analyzed by Western ‐ blot using Phospho ‐ Tyrosine
Mouse mAb (Bioke).
Labeled p27 showed inhibition in Cdk2 activity assays.
Single ‐ molecule Multiparameter
Fluorescence
Detection (smMFD) smMFD for confocal high ‐ precision Förster
Resonance
Energy
Transfer (hpFRET) studies of single ‐ molecules was done using a nm diode laser (LDH ‐ D ‐ C PicoQuant,
Germany, operating at MHz, power at objective µW) exciting freely diffusing labeled molecules that passed through a detection volume of the NA collar (0.17) corrected Olympus objective.
The emitted fluorescence signal was collected through the same objective and spatially filtered using a µm pinhole, to define an effective confocal detection volume. We used a new detection and data registration scheme to measure dead time free species cross correlation functions. For that, the signal was divided into parallel and perpendicular components at two different colors (“green” and “red”) through band pass filters, HQ and HQ for green and red respectively, and split further with beam splitters. In total eight photon ‐ detectors are used, four for green ( ‐ SPAD,
PicoQuant,
Germany) and four for red channels (APD
SPCM ‐ AQR ‐ Perkin
Elmer,
Germany). A time ‐ correlated single ‐ photon counting (TCSPC) module (HydraHarp PicoQuant,
Germany) with
Time ‐ Tagged
Time ‐ Resolved (TTTR) mode and synchronized input channels was used for data registration. For smMFD (smFA and smFRET) measurements, samples were diluted (buffer used was mM HEPES, pH mM NaCl, µM TROLOX) to pM concentration assuring ~1 burst per second. In long measurements, to avoid drying out of the immersion water, an oil immersion liquid with refraction index of water was used (Immersol, Carl
Zeiss
Inc.,
Germany).
NUNC chambers (Lab ‐ Tek, Thermo
Scientific,
Germany) were used with µL sample volume. Standard controls consisted of measuring water to determine the instrument response function (IRF), buffer for background subtraction, and the nM concentration of green and red standard dyes (Rh110 and Rh101) in water solutions for calibration of green and red channels, respectively. To calibrate the detection efficiencies we used a mixture solution of dual ‐ labeled DNA oligonucleotides with known distance separation between donor and acceptor dyes.
Fluorescence analysis of smFA and smFRET experiments smFA and smFRET experiments were analyzed using smMFD (Sisamakis et al., We used Probability
Distribution
Analysis (PDA) (Kalinin et al., Kalinin et al., to determine the anisotropy and distance distributions and their corresponding uncertainties. Details on the analysis are given in Supplemental
Methods.
Filtered
Fluorescence
Correlation
Spectroscopy (fFCS) (Felekyan et al., was used to identify the species ‐ specific interconversion rates in smFRET experiments. Details are given in Supplemental
Methods and
Tables.
The software used to perform the analysis, written in house, can be downloaded from Y74 T187Y88
C40 C54 C75 C93 C110 198 AB Figure 1 igure 2
BC D PO -Src PO -p27-KIDY74F/Y88F-p27-KIDY88F-p27-KID + +pY88-p27-KID + +++p27-KID + + + + + +++Cdk2/cyclin A - - - + + +- -10 -9 -8 -7 -6020406080100 ac t i v i t y [ a . u . ] C d k2 k i n ase p27-KID pY88- p27-KID pY74/pY88- p27-KID Concentration of p27 species[log(M)] A T187 Rb p p Y - p p Y / p Y - p T187 E x t e n t o f s ub s t r a t e pho s pho r y l a t i on [ a . u . ] p p Y - p p Y / p Y - p p107 p27-KID pY88- p27-KID pY74/pY88- p27-KID pho s pho r y l a t i on [ a . u . ] R a t e o f T Concentration of 1:1:1complexes [ M] pY88-p27p27pY74/pY88-p27 igure 3 A p27-KID assignments notavailable pY88-p27-KID Residues83-89assignments notavailable [ pp m ]
30 40 50 60 70 80 900.00.51.01.52.0 pY74/pY88-p27-KID Residues62-89assignments notavailable p27-KID residue number
Cdk2/cyclin
A Cdk2/cyclin
A/p27 ‐ KID ‐ C Cdk2/cyclin
A/p27 ‐ KID B igure 4 Anisotropy
Fractions A
29 40 54 75 930255075100 F r ac t i on s [ % ] Low r ss High r ss No P pY88 pY88 pY74
29 40 54 75 930.00.10.20.30.4 A n i s o t r op y Label position p27 B L = 1.3 ns A n i s o t r op y r D D f [ns] No P pY88 H = 17.6 ns D f [ns] H = 15.3 ns L = 1.9 ns o f bu r s t s t = 2 ms Cdk2/cyclin A/p27 ‐ C93 C F r ac t i on s [ % ] Low R DA High R DA No P pY88 pY88 pY74 R DA [ Å ] Label positions p27 D Cdk2/cyclin
A/p27 ‐ C54 ‐ E R DA E [ Å ] D(A) f [ns] 0 2 4 6 pY88 D(A) f [ns] No P o f bu r s t s t = 3 ms FRET
Fractions -5 -4 -3 -2 -1 -4 -3 -2 -1 Dynamics: global C o rr . a m p li t ud e N o P localchain sACF LF-LF No P HF-HF No P sCCF
HF-LF No P local globalchain sACF
LF-LF pY88 HF-HF pY88 C o rr . a m p li t ud e p Y N o r m . t o N o P sCCF HF-LF pY88 sACF
No P pY88 C o rr . a m p li t ud e F i t s O ve r l ay Correlation time [ms] sACF
No P pY88
Correlation time [ms] igure 5
Dynamic anticipation by Cdk2/Cyclin A ‐ bound p27 mediates signal integration in cell cycle regulation Maksym
Tsytlonok *, Hugo
Sanabria *, Yuefeng
Wang *, Suren
Felekyan , Katherina
Hemmen , Mi ‐ Kyung
Yun , Brett
Waddell , Cheon ‐ Gil
Park , Sivaraja
Vaithiyalingam , Luigi
Iconaru , Stephen W. White , Peter
Tompa **,
Claus A. M. Seidel **, and Richard
Kriwacki ** VIB
Center for
Structural
Biology,
Vrije
Universiteit
Brussel,
Brussels,
Belgium. Department of Physics and
Astronomy,
Clemson
University,
Clemson,
SC,
USA. Lehrstuhl für
Molekulare
Physikalische
Chemie,
Heinrich ‐ Heine ‐ Universität,
Düsseldorf,
Germany. Department of Structural
Biology,
St.
Jude
Children’s
Research
Hospital,
Danny
Thomas
Place,
Memphis, TN USA. Current address:
Department of Radiation
Oncology,
West
Cancer
Center,
University of Tennessee
Health
Sciences
Center,
Memphis, TN USA Molecular
Interaction
Analysis
Shared
Resource,
St.
Jude
Children’s
Research
Hospital,
Danny
Thomas
Place,
Memphis, TN USA. Institute of Enzymology,
Research
Centre for
Natural
Sciences of the Hungarian
Academy of Sciences,
Budapest,
Hungary. Department of Microbiology,
Immunology and
Biochemistry,
University of Tennessee
Health
Sciences
Center,
Memphis, TN USA. *, ** these authors contributed equally to the work **Correspondence: [email protected] (PT), [email protected] (CAMS) and [email protected] (RK) Supplemental
Information
Table of Contents
S1. Supplemental Methods ................................................................................................................ 4
S1.1.
Burst analysis and parametric lines for
FRET and anisotropy multidimensional histograms ............................................................................................................................................ 4
S1.2.
Probability
Distribution
Analysis (PDA) of single ‐ molecule Fluorescence
Anisotropy (smFA) and
Förster
Resonance
Energy
Transfer (smFRET) experiments . 6
S1.3.
Statistical uncertainties in PDA ........................................................................................... 7
S1.4.
Filtered
Fluorescence
Correlation
Spectroscopy .......................................................... 7
S1.5.
Accessible volume (AV) model and inter ‐ fluorophore distances ............................ 9 S2. Supplemental Tables ................................................................................................................. 10 Supplemental
Table Data collection and refinement statistics for determination of the structure of the Cdk2/cyclin
A/p27 ‐ KID ‐ C complex using X ‐ ray crystallography. ................................................................................................................................ 10 Supplemental
Table Fit parameters of smFA experiments obtained by PDA (Sections
S1.2,
S1.3). ...................................................................................................................... 11
Supplemental
Table Brightness correction for smFA experiments. .......................... 12
Supplemental
Table Rotational correlation time for smFA experiments. .............. 13
Supplemental
Table Fluorophore properties of dyes used in smFRET experiments and generated FRET lines according to S1.1 equations (3) and (7). .............................. 14
Supplemental
Table Fit parameters of PDA analysis for smFRET experiments. ... 16
Supplemental
Table F D /F A levels for High and
Low R DA E . ........................................... 17 Supplemental
Table Inter ‐ fluorophore distances of p27 FRET variants in complex with Cdk2/cyclin A based on accessible volume calculations (Section S1.5) and comparison with the “major” state in the non ‐ phosphorylated state. ........................ 18 Supplemental
Table Relaxation rate constants obtained by fFCS for Figure in main text (Section S1.4,
Equations (18) ‐ (20)). ....................................................................... 19 Supplemental
Table
Result of the analysis of triplicate measurements of p27 ‐ KID, pY88 ‐ p27 ‐ KID, and pY74/Y88 ‐ p27 ‐ KID binding to Cdk2/cyclin A, and separately to cyclin A or Cdk2, using surface plasmon resonance (SPR). .......................................... 21 S3. Supplemental Figures ............................................................................................................... 22
Supplemental
Figure Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A activity. ................................................... 22 Supplemental
Figure (next page). Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A and promotes phosphorylation of p27 on T187. .............................................................................................. 23
Supplemental
Figure Ejection of pY88 and the residue ‐ region from the Cdk2 active site is mimicked by truncation of p27 ‐ KID at residue .......................... 25 Supplemental
Figure Multidimensional smFA histograms of p27/Cdk2/cyclin A at various phosphorylation states. ................................................................................................. 26 Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/cyclin A at various phosphorylated states. ............................................................................................. 28 Supplemental
Figure Filtered
FCS
Species auto and cross ‐ correlation (sACF and sCCF) function of smFRET experiments for Cdk2/cyclin
A/p27 samples. .................... 30
Supplemental
Figure Representative results for varied concentrations of Cdk2/cyclin A, cyclin A, or Cdk2 binding to p27 ‐ KID, pY88 ‐ p27 ‐ KID, or pY74/Y88 ‐ p27 ‐ KID separately immobilized on the sensor surface. ................................................... 32 Supplemental
Figure (next page). Representative binding isotherms for injected p27 (A),
Y88E ‐ p27 (B), or Y74E/Y88E ‐ p27 (C) binding to Cdk2/cyclin A at temperatures from °C to °C recorded using isothermal titration calorimetry (ITC). ..................................................................................................................................................... 33 Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/Cyclin A with phosphomimetic variants. .................................................................................................. 34 Supplemental
Figure
The effects of mono and dual tyrosine phosphorylation on regulation of Cdk2 by p27 can be mimicked by mutation of Y88, and
Y74 and
Y88, to glutamate (E). .............................................................................................................................. 36 S1.
Supplemental
Methods
S1.1.
Burst analysis and parametric lines for
FRET and anisotropy multidimensional histograms To identify single ‐ molecule events we use “burstwise” or “time ‐ window” selection with criteria out of the mean background count rate. Cutoff times may vary from sample to sample depending on the background signal. Time ‐ resolved fluorescence histograms of each single ‐ molecule event, with a minimum of photons, is processed and fitted using a maximum likelihood algorithm (Maus et al., in custom developed programs coded in LabVIEW (National
Instruments
Co.).
Fluorescent bursts are plotted in histograms (Origin OriginLab
Co).
For single ‐ molecule Förster
Resonance
Energy
Transfer (smFRET) experiments, bursts are shown using a parametric relationship between the ratio of the donor fluorescence over the acceptor fluorescence ( F D / F A ) and the fluorescence ‐ weighted donor lifetime obtained in burst analysis τ D (A) f . F D / F A depends on specific experimental parameters such as count rate per color channel ( S G and S R ), the fluorescence quantum yields of the dyes ( FD (0) and FA for donor and acceptor respectively), background ( B G and B R for green and red channels), detection efficiencies ( g G and g R for green and red respectively) and crosstalk ( ) following these relationships G GGD g BSF , ( ) R RGRA g BFSF . ( ) In the F D / F A vs. τ D (A) f representations it is useful to represent a static FRET line that represent the parametric relationship between ( F D / F A ) and τ D (A) f which include the dynamics of the fluorophore’s linker. Consequently, the linker flexibility generates a distribution of distances instead of a single distance. Mathematically, the
FRET lines for F D / F A vs. τ D (A) f and R D A vs. τ D (A) f , corrected for linker mobility, are
130 ,)(, )0()0(Lstatic, i iLxADLi DFAFDAD AFF . ( ) DA D ii L D A x Li
R R B . ( ) The L sub index notation is to identify and specify the linker effects and τ D (0) is the donor fluorescence lifetime in the absence of acceptor. The “ A i,L ” and “ B i,L ” coefficients are empirically determined by a polynomial approximation of the following parametric relationship between the species average lifetime τ D (A) x,L and fluorescence weighted average lifetime τ D (A) f,L for a range for R DA =[1 Å to R ] Å using the following relationships DADAADLxAD LxAD DADAADLfADfAD i iLfADLiLxAD dRRp dRRpA )( )( )(,)( ,)(2)(,)()( 30 ,)(,,)( ( ) Here, the distribution of distances is assumed to follow a Gaussian probability function with a mean FRET distance R DA and standard deviation D A , DADADADADA
RRRp . ( ) Keep in mind that in Eq. there is a R DA for each τ D (A) following the Förster relationship DADAD RR , (6) where R is the Förster distance.
For our particular set of dyes R = Å and we assume isotropic reorientation of dyes ( = in the determination of R . The static
FRET line corrected for linker dynamics is only valid for cases where there is no dynamic interexchange between states. In addition to the static FRET line, we use the dynamic FRET line to show the interexchange between states. In this case, a mixed fluorescence species arises from the interconversion between two conformational states, where each state follows the Gaussian distribution stated above.
For the simplest case the dynamic
FRET line can be presented as(Kalinin et al., )0( 2130 )(,21 21)0()0(Ldyn, D ffi ifADLiff ffDFAFDAD
CFF , (7) where D (A) f,L is the mixed fluorescence lifetime, and f and f are two donor fluorescence lifetimes in presence of acceptor corresponding to the states that give rise to the dynamic exchange. The “ C i,L ” coefficients are determined for each FRET pair and differ from the “ A i,L ” coefficients in the static FRET lines.
The L sub index notation is to identify and specify the linker effects. To represent single ‐ molecule Fluorescence
Anisotropy (smFA) we chose the scatter ‐ corrected fluorescence anisotropy per burst ( r D ), which is calculated as (Schaffer et al., lBSlBSG BSBSGr r rD , (8) where G r is the ratio of the detection efficiencies ( g ⊥ / g || ) on the perpendicular ( ⊥) and parallel (||) channels, sometimes referred as G ‐ factor, l = and l = are correction factors for the depolarization created by the microscope objective (Koshioka et al., Schaffer et al., and the signal in the parallel and perpendicular detector are S || and S respectively. In smFA experiments, the parametric histogram used is r D vs. D which relates the steady state anisotropy and the average fluorescence lifetime per burst. In ensemble conditions there is a similar relation called Perrins’
Eq. DD rr , (9) where r is the fundamental anisotropy of the fluorophore and is set to be is the rotational correlation time and D is the average fluorescence lifetime per burst. S1.2.
Probability
Distribution
Analysis (PDA) of single ‐ molecule Fluorescence
Anisotropy (smFA) and
Förster
Resonance
Energy
Transfer (smFRET) experiments To model the shape of the anisotropy and F D / F A distributions, we use probability distribution analysis or PDA.
Anisotropy ‐ PDA is an extension of the FRET ‐ PDA theory which was derived first.
The theory behind these can be found in (Antonik et al., Kalinin et al., In short, the measured fluorescence signal S, consisting of fluorescence ( F ) and background ( B ) photons are expressed in photon count numbers per time window ( t) of a fixed length. In Multiparameter
Fluorescence
Detection the signal is split into two spectral windows termed “green” and “red” each with two polarization components (Parallel “||” and Perpendicular “ ”). The probability of observing a certain combination of photon counts in two detection channels and (e.g., “1=green” and “2=red” or “1=||” and “2= ”) and measured by two or more single photon counting detectors., P ( S , S ), is given by a product of independent probabilities ; 212121 )()()|,()(),( SBFSBF
BPBPFFFPFPSSP . (10) P ( F ) describes the fluorescence intensity distribution, i.e., the probability of observing exactly F fluorescence photons per time window ( t ). P ( B ) and P ( B ) represent the background intensity distributions. )|,( FFFP is the conditional probability of observing a particular combination of F and F , provided the total number of fluorescence photons is F .This can be expressed as )1()!(! !!! !)|,( FFFFF ppFFF FppFF FFFFP . ( p stands for the probability of a detected photon to be registered by the first detector (e.g., green in a FRET experiment or parallel in an anisotropy experiment). For the case of smFA experiments p = p || and it is written as ||21 1|| pplrGGlr lrp rr ( ) where G r , l and l were previously defined. Consequently, p = p . For smFRET p is unambiguously related to the FRET efficiency E according to |12)0(|1 ppGEEp FDFA . ( ) Here, G stands for the ratio of the detection efficiencies in the spectral windows ( G = g G / g R ) and the quantum yields ( FD (0) and FA ) were previously defined. The distribution P ( F ) in Eq. (10) is not directly measurable, instead the total signal intensity distribution P ( S ) is measured, which is given by )()()( BPFPSP , ( ) where P ( B ) is the distribution probability of background counts. Details on the deconvolution procedure are described elsewhere (Kalinin et al., Finally, Eq (10) can be extended for multiple species with the brightness correction used in this work (Kalinin et al., Each species distributions has a half width (hw DA ) which depends mostly on shot noise and photophysical properties of the acceptor fluorophore. S1.3.
Statistical uncertainties in PDA
Confidence intervals estimation for multiple fit parameters is performed as follows. All free fit parameters are varied simultaneously in a random manner. The r value is calculated at random points yielding ‐ points with r values below r N rr ( ) where N is the number of bins and r is the reduced chi ‐ squared value of the best fit. The threshold of r is assigned as confidence interval. One could calculate thresholds.
Alternative methods for threshold determination are available (Soong, however, in practice r is often affected by experimental imperfections and can be considerably larger than one. For this reason, we prefer this test to measure the robustness of the fits providing numerical uncertainties of the free parameters. S1.4.
Filtered
Fluorescence
Correlation
Spectroscopy To separate species, we use filtered FCS (fFCS) (Böhmer et al., Felekyan et al., fFCS differs from standard FCS (Elson and
Magde, and
FRET ‐ FCS (Felekyan et al., by interrogating the “species” (conformational states) fluctuations instead of photon count rates (Felekyan et al., We define the species auto ‐ or cross ‐ correlation function as Ldj cjmjLdj jij Ldj cjmjLdj jijcmi cmicmi ttSftSf ttSftSfttFtF ttFtFtG , (16) where ( i ) and ( m) are two selected “species” in a mixture. When i = m we say it is the species auto ‐ correlation function (sACF), and when i ≠ m it is the species cross ‐ correlation function (sCCF). The difference from standard
FCS is that in fFCS we introduce a set of filters, )( ij f , that depend on the arrival time of each photon after each excitation pulse. The signal S j ( t ), obtained via pulsed excitation is recorded at each j = ... L TCSPC ‐ channel. The signal and filters per detector, d , are stacked in a single array with dimensions Ld for global minimization as previously shown (Felekyan et al., Filters are defined in such a way that the relative “error” difference between the photon count per species ( w ( i ) ) and the weighted histogram jij Hf )( is minimized as defined in Eq. (17). min iLdj jij wHf . (17) where brackets represent time averaging. The requirement is that the decay histogram H j can be expressed as a linear combination of the conditional probability distributions )( ij p , such as )(1 )( ijni ij pwH , with Ldj ij p . Hence, the sCCF provides maximal contrast for intercrossing dynamics (Felekyan et al., One major advantage of sCCF is that if photophysical properties are decoupled from species selection the intercrossing dynamics (Felekyan et al., is recovered with great fidelity. To properly fit the species auto ‐ and cross ‐ correlation function we used a set of equations previously presented (Felekyan et al., BcmiR RcRmimicmidiffCCcmi cmBR RcRmmmTcmmcmdiffBrcmm ciBR RcRiiiTciicidiffBrcii ttBttXCCtGNtG tGttACttTTtGNtG tGttACttTTtGNtG expexp expexp expexp ,)(,,),(, )()( ,)()()(, )()(,)()()(, (18) where t R are the relaxation times that correspond to the exchange times between selected species with corresponding absolute amplitudes of the sACF )(, Rxx AC and the relative normalized amplitudes of the sCCF )(, Rxx CC . )( x T is the triplet amplitude, however, triplet states dynamic was not found in the measured samples. N br is the number of bright molecules in the sACF’s in the focus and N CC of the sCCF’s corresponds to the inverse of the initial amplitude mi G , . cxB tG )( is defined for bleaching term: BcxxcxB ttBBtG exp1 )()()( , (19) cxdiff tG )( is the diffusion term of species x : xdiffcxdiffccxdiff ttztttG . (20) A ‐ dimensional Gaussian shaped volume element parameters ω and z is considered. We assume that cmdiffcidiffcdiff tGtGtG )()( take the form of Eq. (20).In fFCS the amplitudes are highly dependent on the brightness of the individual sub ‐ states and the exchange rate constants. Thus a direct interpretation is not straightforward (Felekyan et al., S1.5.
Accessible volume (AV) model and inter ‐ fluorophore distances To accurately compare FRET ‐ derived distances with structural information provided by crystallography data it is imperative to consider the dimensions of the fluorophores. To do so, we compute the accessible volume of the dyes by considering them as hard sphere models connected to the protein via flexible linkers (modeled as a flexible cylindrical pipe) (Sindbert et al., The overall dimension (width and length) of the linker is based on their chemical structures. For
Alexa maleimide the five carbon linker length was set to Å, the width of the linker is Å and a three sphere model was used to model the dye R = Å, R = Å and R = Å. For
Alexa maleimide the dimensions used were: length = Å, width = Å and the dye radii R = Å, R = Å and R = Å. To account for dye linker mobility we generated a series of AV’s for donor and acceptor dyes attached to p27 placing the dyes at multiple separation distances. For each pair of AV’s, we calculated the distance between dye mean positions ( R mp ) ni mj jAiDjAiDmp RmRnRRR , (21) where )( iD R and )( iA R are all the possible positions that the donor fluorophore and the acceptor fluorophore can take. However, in single ‐ molecule FRET experiment where a ratiometric FRET is calculated the distances is weighted by the average fluorescence thus the mean donor ‐ acceptor distance observed is ERR
EDA (22) where the average efficiency is defined as ni mj jAiD RRR RnmE . S2.
Supplemental
Tables
Supplemental
Table Data collection and refinement statistics for determination of the structure of the Cdk2/cyclin
A/p27 ‐ KID ‐ C complex using X ‐ ray crystallography. The structure of the Cdk2/cyclin
A/p27 ‐ KID ‐ C complex has been deposited and validated bythe PDB with the file name for release upon publication.
Cdk2/cyclin
A/p27 ‐ KID ‐ C Data
Collection a Space
Group P2 Cell dimensions a , b , c (Å) , , No. of crystals (Å) (Å) ‐ (1.89 ‐ unique reflections (6,848) R merge b (0.522)Completeness (%) (96.5)Redundancy (4.2) I / (2.1) Refinement
Resolution (Å) ‐ of reflections R work / R free c atoms Protein
Ion Water B ‐ factor (Å ) deviations Bond lengths (Å)
Bond angles ( ) Ramachandran plot
Favored (%)
Allowed (%)
Outliers (%) a Values in parenthesis are for highest ‐ resolution shell. b IIIR merge , where I is the observed intensity. c R free is the R value obtained for a test set of reflections consisting of randomly selected subset of the data set excluded from refinement. Supplemental
Table Fit parameters of smFA experiments obtained by PDA (Sections
S1.2,
S1.3) . Residue number
Anisotropy (Fraction (%)) No phosphorylation r2 Anisotropy (Fraction (%)) pY88 r2 Anisotropy (Fraction (%)) pY74/pY88 r2 Low r D High r D Low r D High r D Low r D High r D (12.9 (87.1 (25.6 (74.4 (26.7 (73.3 (57.7 (42.3 (62.0 (38.0 (61.8 (38.2 (30.8 (69.2 (29.9 (70.1 (71.7 (28.3 (27.2 (72.8 (25.4 (74.6 (40.5 (59.5 (33.3 (66.7 (65.9 (34.1 (65.1 (34.9 Supplemental
Table Brightness correction for smFA experiments.
Normalized to the maximum lifetime: Q (short lifetime) = D (short) / D (long) . Sample in complex D (High rD) [ns] D (Low rD) [ns] Q (High rD Q (Low rD) p27 C29 No phosphorylation ‐ p27 C29 ‐ p27 C29
C40 No phosphorylation ‐ p27 C40 ‐ p27 C40
C54 No phosphorylation ‐ p27 C54 ‐ p27 C54
C75 No phosphorylation ‐ p27 C75 ‐ p27 C75
C93 No phosphorylation ‐ p27 C93 ‐ p27 C93 Supplemental
Table Rotational correlation time for smFA experiments.
Values were obtained using
Table Table and Perrin’s
Equation [Eq. (9)] for
Cdk2/cyclin
A/p27 samples.
Rotational
Correlation (ns) No phosphorylation pY88 pY74/pY88 Residue
No.
Low r D High r D Low r D High r D Low r D High r D Supplemental
Table Fluorophore properties of dyes used in smFRET experiments and generated FRET lines according to S1.1 equations (3) and (7).
Sample in complex D(0) A Static
FRET
LineDynamic
FRET
Line p27
C29/54 No phosphorylation (0.6988/0.39)/((3.7494/(( ‐ D(A) f3 )+(0.3131* D(A) f2 )+0.5690* D(A) f + ‐ ‐ ‐ (1.4769* D(A) f + ‐ ‐ pY88 ‐ p27 C29/54 (0.7220/0.36)/((3.9015/(( ‐ D(A) f3 )+(0.3002* D(A) f2 )+0.5507* D(A) f + ‐ ‐ ‐ (1.5937* D(A) f + ‐ ‐ pY74/pY88 ‐ p27 C29/54 (0.7239/0.37)/((4.0239/(( ‐ D(A) f3 )+(0.2978* D(A) f2 )+0.5875* D(A) f + ‐ ‐ ‐ (1.5244* D(A) f + ‐ ‐ E88 ‐ p27 C29/54 (0.6036/0.37)/((3.511/(( ‐ D(A) f3 )+(0.2965* D(A) f2 )+0.6838*x+ ‐ ‐ E74/E88 ‐ p27 C29/54 (0.5948/0.394)/((3.5383/(( ‐ D(A) f3 )+(0.282* D(A) f2 )+0.7161* D(A) f + ‐ ‐ p27 C54/93 No phosphorylation (0.7194/0.42)/((3.8301/(( ‐ D(A) f3 )+(0.3112* D(A) f2 )+0.5553* D(A) f + ‐ ‐ ‐ (1.3896* D(A) f + ‐ ‐ pY88 ‐ p27 C54/93 (0.7519/0.37)/((3.9997/(( ‐ D(A) f3 )+(0.2947* D(A) f2 )+0.5367* D(A) f + ‐ ‐ ‐ (1.4414* D(A) f + ‐ ‐ pY74/pY88 ‐ p27 C54/93 (0.7541/0.36)/((3.9983/(( ‐ D(A) f3 )+(0.3068* D(A) f2 )+0.5289* D(A) f + ‐ ‐ ‐ (1.4602* D(A) f + ‐ ‐ E88 ‐ p27 C54/93 (0.6036/0.363)/((3.511/(( ‐ D(A) f3 )+(0.2965* D(A) f2 )+0.6838* D(A) f + ‐ ‐ E74/E88 ‐ p27 C54/93 (0.683/0.33)/((3.7855/(( ‐ D(A) f3 )+(0.2846* D(A) f2 )+0.6179* D(A) f + ‐ ‐ p27 C75/110 No phosphorylation (0.6324/0.39)/((3.7203/(( ‐ D(A) f3 )+(0.2782* D(A) f2 )+0.6777* D(A) f + ‐ ‐ ‐ (1.3731* D(A) f + ‐ ‐ pY88 ‐ p27 C75/110 (0.6332/0.36)/((3.7901/(( ‐ D(A) f3 )+(0.2612* D(A) f2 )+0.7019* D(A) f + ‐ ‐ ‐ (1.1605* D(A) f + ‐ ‐ pY74/pY88 ‐ p27 C75/110 (0.6025/0.37)/((3.7301/(( ‐ D(A) f3 )+(0.2502* D(A) f2 )+0.7510* D(A) f + ‐ ‐ ‐ (1.1961* D(A) f + ‐ ‐ E88 ‐ p27 C75/110 (0.7129/0.385)/((3.8577/(( ‐ D(A) f3 )+(0.2868* D(A) f2 )+0.5847* D(A) f + ‐ ‐ E74/E88 ‐ p27 C75/110 (0.6482/0.375)/((3.7123/(( ‐ D(A) f3 )+(0.2802* D(A) f2 )+0.6602* D(A) f + ‐ ‐ Supplemental
Table Fit parameters of PDA analysis for smFRET experiments.
Global fitting Δ T=1, and ms. is shown for Δ T=3 ms. A) All fractions B) Renormalized fractions for only
FRET species.
Half ‐ width distribution (hw DA ) of the PDA distribution should not be confused with σ DA in Eq. (5).
Data was corrected as described in S1.2 and using values from
Table A) Samples
Cdk2/cyclin
A/p27 R DA E [Å] (Fraction (%)) hw DA [Å] No P r2 R DA E [Å] (Fraction (%)) hw DA [Å] pY88 r2 R DA E [Å] (Fraction (%)) hw DA [Å] pY74/pY88 r2 Low R DA E High R DA E Dirt D only Low R DA E High R DA E Dirt D only Low R DA E High R DA E Dirt D only C29 ‐ (11.1 (59.9 (22.8) (6.2) ‐‐ (9.2 (30.0 (48.6) (12.2) ‐‐ (14.4 (22.2 (44.3)17.6 (19.1) ‐‐ C54 ‐ (55.7 (11.6 (24.7) (8.0) ‐‐ (20.2 (16.9 (52.9) (10.0) ‐‐ (18.9 (7.4 (44.7)21.2 (29.0) ‐‐ C75 ‐ (6.4 (37.1 (43.1) (13.5) ‐‐ (17.8 (13.2 (63.7) (5.2) ‐‐ (23.3 (15.4 (37.5)18.1 (23.9) ‐‐ B) Samples
Cdk2/cyclin
A/p27 No P Fraction (%) pY88
Fraction (%) pY74/pY88
Fraction (%)
Low R DA E High R DA E Low R DA E High R DA E Low R DA E High R DA E C29 ‐ C54 ‐ C75 ‐ Supplemental
Table F D /F A levels for High and
Low R DA E . R DA E were converted to FD/FA with
Eq. (6) and (3) (Table
Sample in ternary complex F D /F A ( R DA E(Low) ) F D /F A ( R DA E(High) ) p27 C29/54 No P ‐ p27 C29/54 ‐ p27 C29/54 0.52 2.04E88 ‐ p27 C29/54
E74/E88 ‐ p27 C29/54 p27
C54/93 No P ‐ p27 C54/93 ‐ p27 C54/93 0.99 2.27E88 ‐ p27 C54/93
E74/E88 ‐ p27 C54/93 p27
C75/110 No P ‐ p27 C75/110 ‐ p27 C75/110 1.1 1.42E88 ‐ p27 C75/110 ‐ p27 C75/110 Supplemental
Table Inter ‐ fluorophore distances of p27 FRET variants in complex with Cdk2/cyclin A based on accessible volume calculations (Section S1.5) and comparison with the “major” state in the non ‐ phosphorylated state. Sample Å Cdk2/cyclin
A/p27
Fig . pdb:1JSU Å Cdk2/cyclin
A/p27 ‐ C Å Experiment Å R DA E C ‐ C R DA E C ‐ C R DA E C ‐ C R DA E C29 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ Supplemental
Table Relaxation rate constants obtained by fFCS for Figure in main text (Section S1.4,
Equations (18) ‐ (20)). Correlation b N CC /N Br t diff [ms] ω CC t R1 [µs] X /AC t R2 [µs] X /AC t R3 [µs] X /AC t R4 [µs] X /AC B t B [ms] p27 C29
C54 in complex with Cdk2/cyclin A No P LF ‐ HF HF ‐ LF ‐ LF ‐ HF LF ‐ HF HF ‐ LF ‐ LF ‐ HF ‐ pY74 LF ‐ HF HF ‐ LF ‐ LF ‐ HF p27 C54
C93 in complex with Cdk2/cyclin A No P LF ‐ HF HF ‐ LF ‐ LF ‐ HF LF ‐ HF HF ‐ LF ‐ LF ‐ HF ‐ pY74 LF ‐ HF HF ‐ LF ‐ LF HF ‐ HF p27 C75
C110 in complex with Cdk2/cyclin A No P LF ‐ HF HF ‐ LF ‐ LF ‐ HF LF ‐ HF HF ‐ LF ‐ LF ‐ HF ‐ pY74 LF ‐ HF HF ‐ LF ‐ LF ‐ HF Supplemental
Table
Result of the analysis of triplicate measurements of p27 ‐ KID, pY88 ‐ p27 ‐ KID, and pY74/Y88 ‐ p27 ‐ KID binding to Cdk2/cyclin A, and separately to cyclin A or Cdk2, using surface plasmon resonance (SPR).
The data were fit to a Langmuir interaction model.
Interacting species k a (M ‐ s ‐ ) k d (s ‐ ) K D (nM) Rmax (RU) p27 ‐ KID + Cdk2/cyclin A ± x ± x ‐ ± ± ‐ p27 ‐ KID + Cdk2/cyclin A ± x ± x ‐ ± ± ‐ p27 ‐ KID + Cdk2/cyclin A ± x ± x ‐ ± ± ‐ KID + cyclin A ± x ± x ‐ ± ± ‐ p27 ‐ KID + cyclin A ± x ± x ‐ ± ± ‐ p27 ‐ KID + cyclin A ± x ± x ‐ ± ± ‐ KID + Cdk2 ± x ± x ‐ ± ± ‐ p27 ‐ KID + Cdk2 ± x ± x ‐ ± ± ‐ p27 ‐ KID + Cdk2 ± x ± x ‐ ± ± S3.
Supplemental
Figures
Supplemental
Figure Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A activity. (A) Representative results of kinase activity assays for Cdk2/cyclin A in the presence of increasing concentrations of unphosphorylated and mono and dual Y phosphorylated p27 ‐ KID and p27.
Autoradiography was used to monitor incorporation of P ‐ labeled phosphate into the substrate, Histone
H1, which was resolved using
SDS ‐ PAGE.
The regions of the autoradiograms corresponding to the Histone H1 protein in the gels are shown. The experiments were performed in triplicate. The results for p27 ‐ KID, pY88 ‐ p27 ‐ KID and pY74/pY88 ‐ p27 ‐ KID are quantified in Fig. as average kinase activity (± standard deviation of the mean) relative to that in the absence of p27 ‐ KID. (B)
Quantification of the results presented in (A) for mono and dual Y phosphorylated p27. The results are quantified as in Fig. A B Supplemental
Figure (next page). Incremental phosphorylation of tyrosine residues in p27 exerts rheostat ‐ like control over Cdk2/cyclin A and promotes phosphorylation of p27 on T187. (A)
Representative results of kinase activity assays for different concentrations of complexes of Cdk2/cyclin A with p27, pY88 ‐ p27 or pY74/pY88 ‐ p27 with T187 (of p27) as the substrate. Autoradiography was used to monitor incorporation of P ‐ labeled phosphate into T187 of p27, which was resolved using SDS ‐ PAGE.
The regions of the autoradiograms corresponding to the p27 protein in the gels are shown. The experiments were performed in triplicate. These results are quantified in Fig. as average kinase activity (± standard deviation of the mean) relative to that for the highest concentration of pY74/pY88 ‐ p27/Cdk2/cyclin A. (B) Representative results of kinase activity assays for and μ M complexes of Cdk2/cyclin A with p27, pY88 ‐ p27 or pY74/pY88 ‐ p27. In addition, equimolar amounts of either Nus ‐ tagged Rb C ‐ terminus (Nus ‐ Rb) or GST/His ‐ tagged p107 C ‐ terminus (p107) were included as “in trans” Cdk2 substrates.
Autoradiography was used to monitor incorporation of P ‐ labeled phosphate into T187 of p27 and Nus ‐ Rb or p107, which were resolved using SDS ‐ PAGE.
The regions of the autoradiograms corresponding to the p27 and Nus ‐ Rb or p107 proteins in the gels are shown. The experiments were performed in triplicate. The results for the μ M complexes and Nus ‐ Rb or p107 are quantified in Fig. as average kinase activity (± standard deviation of the mean) relative to that for P ‐ labeled Nus ‐ Rb. A B C Supplemental
Figure Ejection of pY88 and the residue ‐ region from the Cdk2 active site is mimicked by truncation of p27 ‐ KID at residue (A) Left, representative results of kinase activity assays for Cdk2/cyclin A in the presence of increasing concentrations of p27 ‐ KID, pY88 ‐ p27 ‐ KID or p27 ‐ KID ‐Δ C (p27 ‐ KID with residues ‐ deleted). Autoradiography was used to monitor incorporation of P ‐ labeled phosphate into the substrate, Histone
H1, which was resolved using
SDS ‐ PAGE.
The regions of the autoradiograms corresponding to the Histone H1 protein in the gels are shown. The experiments were performed in triplicate. Right, quantification of the results on the left showing average kinase activity (± standard deviation of the mean) relative to that for pM p27 ‐ KID. (B)
Superposition of the structures of Cdk2/cyclin A bound to ATP (PDB:1JST;
Cdk2/cyclin
A:ATP), p27 ‐ KID bound to Cdk2/cyclin A (PDB:1JSU; p27 ‐ KID/Cdk2/cyclin A) and p27 ‐ KID ‐ C bound to Cdk2/cyclin A (p27 ‐ KID ‐ Δ
C/Cdk2/cyclin A, determined in this study). The structures are superimposed on backbone heavy atoms of cyclin A. The color code is indicated in the illustration and the boxed region is illustrated in Fig.
The
PyMOL
Molecular
Graphics
System (Schrödinger,
LLC) was used to prepare the illustration. A B Supplemental
Figure Multidimensional smFA histograms of p27/Cdk2/cyclin A at various phosphorylation states. Two ‐ dimensional histogram of scatter anisotropy (r D ) vs. D(A) f for Bodipy labeled single
Cys p27 variants in complex with Cdk2/Cyclin A. “Burstwise” analysis of A) C29, B) C40, C) C54, D) C75 and E) C93 variants.
For all cases one dimensional projections for D ( A ) f and anisotropy are also shown. Pure donor fluorescence ( F D ) is corrected for background ( B G = kHz,). Perrin’s equation for r low (blue) and r high (purple) using )r H( ighD and )r Low( D are shown (Suppl. Table and Suppl.
Table
Rotational correlation times ρ (L ow rD ) and ρ (High rD) are given in the respective plot. A Cdk2/cyclin
A/p27 ‐ C29 r D D(0) f [ns] No P pY88 pY74/pY88 D(0) f [ns] o f bu r s t s D(0) f [ns] B Cdk2/cyclin
A/p27 ‐ C40 r D D(0) f [ns] No P pY88 pY74/pY88 D(0) f [ns] o f bu r s t s D(0) f [ns] C Cdk2/cyclin
A/p27 ‐ C54 r D D(0) f [ns] No P pY88 pY74/pY88 D(0) f [ns] o f bu r s t s D(0) f [ns] D Cdk2/cyclin
A/p27 ‐ C75 r D D(0) f [ns] No P pY88 pY74/pY88 D(0) f [ns] o f bu r s t s D(0) f [ns] E Cdk2/cyclin
A/p27 ‐ C93 r D D(0) f [ns] No P pY88 pY74/pY88 D(0) f [ns] o f bu r s t s D(0) f [ns] Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/cyclin A at various phosphorylated states. Two ‐ dimensional histogram F D / F A vs. lifetime of donor in the presence of acceptor D(A) f , and scatter corrected donor anisotropy (r D ) vs. D(A) f for Cdk2/cyclin
A/p27 with donor and acceptor dyes at various positions in “burstwise” mode. A) C29 ‐ B) C54 ‐ and C) C75 ‐ For all cases one dimensional projections for F D / F A , D ( A ) f and anisotropy are also shown. Pure donor and acceptor fluorescence ( F D and F A ) are corrected for background ( B G = kHz A, B) or kHz for C), B R = kHz A,B) or kHz C), spectral cross ‐ talk ( = %) and detection efficiency ratio (g G /g R = Static
FRET lines [Eq. (3)] are shown in blue. Dynamic
FRET lines [Eq. (7)] between the
Low and
High R DA E states (Suppl. Tables ‐ are shown in magenta. Light and dark horizontal lines mark the F D / F A ratio corresponding to the Low and
High R DA E states. Perrin’s equation with rotational correlation time indicated in the plot is shown as blue line. A Cdk2/cyclin
A/p27 ‐ C29 ‐ -2 -1 F D / F A pY74/pY88pY88 D(A) f [ns] r D No P =3.1ns o f bu r s t s =2.1ns D(A) f [ns] 10000 =1.8ns D(A) f [ns] 600 B Cdk2/cyclin
A/p27 ‐ C54 ‐ -2 -1 F D / F A pY74/pY88pY88 D(A) f [ns] r D No P =3.1ns o f bu r s t s =2.5ns D(A) f [ns] 700 =2.0ns D(A) f [ns] 1700 C Cdk2/cyclin
A/p27 ‐ C75 ‐ -2 -1 F D / F A pY74/pY88pY88 D(A) f [ns] r D No P =3.1ns o f bu r s t s =2.4ns D(A) f [ns] 10000 =1.8ns D(A) f [ns] 500 Supplemental
Figure Filtered
FCS
Species auto and cross ‐ correlation (sACF and sCCF) function of smFRET experiments for Cdk2/cyclin
A/p27 samples.
Filtered
Fluorescence
Auto and
Cross ‐ Correlation, left sACF and right sCCF, respectively.
Filters were selected by “burstwise” selection based on FD/FA arbitrary cutoffs to select low ‐ FRET (LF) or high ‐ FRET (HF) populations.
The integrated fluorescence of these burst corresponds to two independent species. The two sCCF (right) (HF to LF and LF to HF) and the sACF where globally fit using
Eq. (18) to determine the number of relaxation times and their amplitudes (Suppl. Table
Residuals of the fit for the sACF and sCCF are shown on top of each correlation curve. Similar treatment was used for the pY88, data not shown. p27 No P p27 pY88 -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -606 global dynamicslocal dynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -606 chaindynamics local dynamics global dynamics -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -20020 global dynamicslocaldynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -20020 chaindynamics local dynamics global dynamics p27 pY88 pY74 p27 No P -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -20020 global dynamicslocal dynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -20020 chaindynamics local dynamics global dynamics -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -12012 global dynamicslocaldynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -12012 chaindynamics local dynamics global dynamics p27 pY88 p27 pY88 pY74 -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -12012 global dynamicslocal dynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -12012 chaindynamics local dynamics global dynamics -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -30030 global dynamicslocal dynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -30030 chaindynamics local dynamics global dynamics p27 No P p27 pY88 -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -30030 global dynamicslocal dynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -30030 chaindynamics localdynamics global dynamics -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -30030 global dynamicslocaldynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -30030 chaindynamics localdynamics global dynamics p27 pY88 pY74 f r ac t i on No P p Y88 p Y88 pY74 p27 29C/54C p27 54C/93C f r ac t i on p27 75C/110C f r ac t i on Correlation time t c [ms] -5 -4 -3 -2 -1 -4 -3 -2 -1 LF-LF Fit LF-LF HF-HF Fit HF-HF c o rr e l a t i on a m p li t u t ud e Correlation time, [ms] -30030 global dynamicslocaldynamics r es i d . chaindynamics Correlation time, [ms]
LF-HF Fit LF-HF HF-LF Fit HF-LF -30030 chaindynamics localdynamics global dynamics Supplemental
Figure Representative results for varied concentrations of Cdk2/cyclin A, cyclin A, or Cdk2 binding to p27 ‐ KID, pY88 ‐ p27 ‐ KID, or pY74/Y88 ‐ p27 ‐ KID separately immobilized on the sensor surface. The results of triplicate injections are shown with fits of a Langmuir interaction model shown as solid orange curves. The kinetic constants and K D values derived from these analyses are provided in Supplemental
Table p27 pY88 ‐ p27 pY74/pY88 ‐ p27A Binding to:
Cdk2/ cyclin A B Binding to:
Cyclin A C Binding to:
Cdk2
Time (s)
Time (s) Time (s) Supplemental
Figure (next page). Representative binding isotherms for injected p27 (A),
Y88E ‐ p27 (B), or Y74E/Y88E ‐ p27 (C) binding to Cdk2/cyclin A at temperatures from °C to °C recorded using isothermal titration calorimetry (ITC). For each experiment, the upper panel shows the power and the lower panel the heat associated with each injection.
The enthalpy of binding ( Δ H) values derived from analysis of triplicate measurements using a binding model are provided in Table Heat capacity change for binding ( Δ C p ) values were determined from the slope of Δ H versus temperature plots (D); Δ C p values are provided in Table C C C C C A B C D E F
Temperature (°C) Supplemental
Figure Multidimensional smFRET histograms of p27/Cdk2/Cyclin A with phosphomimetic variants. Two ‐ dimensional histogram F D / F A vs. lifetime of donor in the presence of acceptor D(A) f , and scatter corrected donor anisotropy (r D ) vs. D(A) f for Cdk2/Cyclin
A/p27 with donor and acceptor dyes at various positions with phosphomimetic mutations at positions E88 and
E74/88. “Burstwise” mode of A) C29 ‐ B) C54 ‐ and C) C75 ‐ samples. For all cases one dimensional projections for F D / F A , D ( A ) f and anisotropy are also shown. Pure donor and acceptor fluorescence ( F D and F A ) are corrected for background ( B G = kHz, B R = kHz), spectral cross ‐ talk ( = direct acceptor excitation ( β = %) and detection efficiency ratio (g G /g R = Values for “No P” samples are given in the legend to Figure
S5.
Static
FRET lines (Eq. (3)) are shown in blue. Dynamic
FRET lines (Eq. (7)) between the
Low and
High R DA E states (Table are shown in magenta. Perrin’s equation with rotational correlation time indicated in the plot is shown as blue line. A Cdk2/cyclin
A/p27 ‐ C29 ‐ -2 -1 F D / F A E74/E88E88 D(A) f [ns] r D No P D(A) f [ns] D(A) f [ns] =3.1ns o f bu r s t s =1.5ns =2.0ns B Cdk2/cyclin
A/p27 ‐ C54 ‐ -2 -1 F D / F A E74/E88E88 D(A) f [ns] r D No P D(A) f [ns] D(A) f [ns] =3.1ns o f bu r s t s =1.8ns =2.8ns C Cdk2/cyclin
A/p27 ‐ C75 ‐ -2 -1 F D / F A E74/E88E88 D(A) f [ns] r D No P D(A) f [ns] D(A) f [ns] =3.1ns o f bu r s t s =2.5ns =1.1ns Supplemental
Figure
The effects of mono and dual tyrosine phosphorylation on regulation of Cdk2 by p27 can be mimicked by mutation of Y88, and
Y74 and
Y88, to glutamate (E). (A) Results of kinase activity assays for Cdk2/cyclin A in the presence of increasing concentrations of p27, Y88E ‐ p27 or Y74E/Y88E ‐ p27. Autoradiography was used to quantify incorporation of P ‐ labeled phosphate into the substrate, Histone
H1, which was resolved using
SDS ‐ PAGE (not shown).
The experiments were performed in triplicate and the results quantified as average kinase activity (± standard deviation of the mean) relative to that in the presence of the lowest concentration of p27 expressed as percentage activity values. (B) NMR analysis of the influence of tyrosine to glutamate mutagenesis on interactions between p27 ‐ KID and
Cdk2/cyclin A. Chemical shift differences for residues in unmutated (top) and tyrosine to glutamate mutated (Y88E ‐ p27 ‐ KID, middle; and
Y74E/Y88E ‐ p27 ‐ KID, bottom) p27 ‐ KID bound to Cdk2/cyclin A. Residues near
Y88 and within the entire D2 subdomain adopt free state ‐ like conformations in Y88E ‐ p27 ‐ KID and
Y74E/Y88E ‐ p27 ‐ KID, respectively. values were calculated using the equation: = [( H N ) + x ( N H ) ] . A B Supplemental references
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