Qilu Ye

The complex structure of calmodulin bound to a calcineurin peptide.

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25‐residue peptide taken from the CaMBD, joined by a 5‐glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25‐residue CaMBD peptide without the glycine linker at a resolution of 2.1 Å. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N‐lobe from one molecule and C‐lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully‐designed CaM‐sepharose pull‐down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg‐408, Val‐409, and Phe‐410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N‐ and C‐lobes, ultimately keeping these lobes apart and forcing “head‐to‐tail” dimerization to attain the requisite N‐ and C‐lobe pairing for CaMBD binding. Proteins 2008.

Crystal structure of the alpha-kinase domain of Dictyostelium myosin heavy chain kinase A.

Structural analysis identifies features of atypical serine-threonine kinases that may account for their different activities relative to those of conventional kinases. In the Loop Conventional serine-threonine protein kinases constitute a huge family of enzymes that regulate many processes in the cell. In contrast, the family of α-kinases is far smaller and consists of atypical kinases that regulate a much more restricted range of functions. Ye et al. have now solved the crystal structure of a prototypical member of the α-kinase family and identified features of the active site of these enzymes that may account for their different activities relative to those of the conventional kinases. Ye et al. solved the crystal structures of the catalytic domain of Dictyostelium myosin II heavy chain kinase A (MHCK A) bound to various nucleotides and compared them to structures of the kinase domains of TRPM7, another α-kinase, and PKA, a conventional protein kinase. In addition to demonstrating that MHCK A and TRPM7 share an almost identical core catalytic domain, solving these structures has identified critical features that distinguish the α-kinases from the conventional protein kinases. In particular, the authors identified a metal ion-binding loop that regulates access to the active site and an aspartylphosphate residue that may act as an intermediate in the phosphorylation of substrates. Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A) disrupts the assembly and cellular activity of bipolar filaments of myosin II by phosphorylating sites within its α-helical, coiled-coil tail. MHCK A is a member of the atypical α-kinase family of serine and threonine protein kinases and displays no sequence homology to typical eukaryotic protein kinases. We report the crystal structure of the α-kinase domain (A-CAT) of MHCK A. When crystallized in the presence of adenosine triphosphate (ATP), A-CAT contained adenosine monophosphate (AMP) at the active site. However, when crystallized in the presence of ATP and a peptide substrate, which does not appear in the structure, adenosine diphosphate (ADP) was found at the active site and an invariant aspartic acid residue (Asp766) at the active site was phosphorylated. The aspartylphosphate group was exposed to the solvent within an active-site pocket that might function as a docking site for substrates. Access to the aspartylphosphate was regulated by a conformational switch in a loop that bound to a magnesium ion (Mg2+), providing a mechanism that allows α-kinases to sense and respond to local changes in Mg2+.

Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCK A (residues 552–841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806–841). The key residue in the C-tail was identified as Thr825, which was found to be constitutively autophosphorylated. Dephosphorylation of Thr825 using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr825 could be rescued by Pi, phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a Pi-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the Pi-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr825 activates ACAT by providing a covalently tethered ligand for the Pi-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr825 to dock intramolecularly into the Pi-pocket. Allosteric activation is predicted to involve a conformational change in Arg734, which bridges the bound Pi to Asp762 in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.

High‐resolution crystal structure of apolipoprotein(a) kringle IV type 7: Insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV1 to KIV10) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV10 represents the high‐affinity lysine‐binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV5–KIV8 and has been directly demonstrated in KIV7. The present study describes the first crystal structure of apo(a) KIV7, refined to a resolution of 1.45 Å, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr‐62 in KIV7 for the corresponding Phe‐62 residue in KIV10, in conjunction with the presence of Arg‐35 in KIV7, results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg‐35, Tyr‐62, Asp‐54) and a peripheral tyrosine residue (Tyr‐40). These interactions restrain the flexibility of key LBS residues (Arg‐35, Asp‐54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr‐62, as well as the elimination of critical ligand‐stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine‐binding affinity exhibited by KIV7 relative to that of KIV10.

Structural basis of calcineurin activation by calmodulin.

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily.

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo‐keto reductase (AKR) superfamily that is induced by the aldehyde‐containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268:5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 Å resolution. Similar to aldose reductase and other AKRs, CHO reductase is an α/β TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR‐1 (mouse fibroblast growth factor‐regulated protein), especially around the variable C‐terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis. Proteins 2000;38:41–48. ©2000 Wiley‐Liss, Inc.

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

Crystal structure of an aldehyde reductase Y50F mutant‐NADP complex and its implications for substrate binding

Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 Å. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild‐type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis. Proteins 2001;44:12–19.

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

Background: Myosin II heavy chain kinase A (MHCK-A) is a member of the atypical α-kinase family. Results: The catalytic and nucleotide binding properties of the MHCK-A kinase domain are characterized. Conclusion: α-Kinases convert ATP to adenosine via formation of an aspartyl phosphate intermediate. Significance: The α-kinase active site has unique functions that may aid in the design of specific inhibitors. The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min−1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.

Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation

The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.