Julie D. Forman-Kay

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

SummaryThe backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.

Sensitivity of secondary structure propensities to sequence differences between α‐ and γ‐synuclein: Implications for fibrillation

The synucleins are a family of intrinsically disordered proteins involved in various human diseases. α‐Synuclein has been extensively characterized due to its role in Parkinsons disease where it forms intracellular aggregates, while γ‐synuclein is overexpressed in a majority of late‐stage breast cancers. Despite fairly strong sequence similarity between the amyloid‐forming regions of α‐ and γ‐synuclein, γ‐synuclein has only a weak propensity to form amyloid fibrils. We hypothesize that the different fibrillation tendencies of α‐ and γ‐synuclein may be related to differences in structural propensities. Here we have measured chemical shifts for γ‐synuclein and compared them to previously published shifts for α‐synuclein. In order to facilitate direct comparison, we have implemented a simple new technique for re‐referencing chemical shifts that we have found to be highly effective for both disordered and folded proteins. In addition, we have developed a new method that combines different chemical shifts into a single residue‐specific secondary structure propensity (SSP) score. We observe significant differences between α‐ and γ‐synuclein secondary structure propensities. Most interestingly, γ‐synuclein has an increased α‐helical propensity in the amyloid‐forming region that is critical for α‐synuclein fibrillation, suggesting that increased structural stability in this region may protect against γ‐synuclein aggregation. This comparison of residue‐specific secondary structure propensities between intrinsically disordered homologs highlights the sensitivity of transient structure to sequence changes, which we suggest may have been exploited as an evolutionary mechanism for fast modulation of protein structure and, hence, function.

Protein dynamics and conformational disorder in molecular recognition

Recognition requires protein flexibility because it facilitates conformational rearrangements and induced‐fit mechanisms upon target binding. Intrinsic disorder is an extreme on the continuous spectrum of possible protein dynamics and its role in recognition may seem counterintuitive. However, conformational disorder is widely found in many eukaryotic regulatory proteins involved in processes such as signal transduction and transcription. Disordered protein regions may in fact confer advantages over folded proteins in binding. Rapidly interconverting and diverse conformers may create mean electrostatic fields instead of presenting discrete charges. The resultant “polyelectrostatic” interactions allow for the utilization of post‐translational modifications as a means to change the net charge and thereby modify the electrostatic interaction of a disordered region. Plasticity of disordered protein states enables steric advantages over folded proteins and allows for unique binding configurations. Disorder may also have evolutionary advantages, as it facilitates alternative splicing, domain shuffling and protein modularity. As proteins exist in a continuous spectrum of disorder, so do their complexes. Indeed, disordered regions in complexes may control the degree of motion between domains, mask binding sites, be targets of post‐translational modifications, permit overlapping binding motifs, and enable transient binding of different binding partners, making them excellent candidates for signal integrators and explaining their prevalence in eukaryotic signaling pathways. “Dynamic” complexes arise if more than two transient protein interfaces are involved in complex formation of two binding partners in a dynamic equilibrium. “Disordered” complexes, in contrast, do not involve significant ordering of interacting protein segments but rely exclusively on transient contacts. The nature of these interactions is not well understood yet but advancements in the structural characterization of disordered states will help us gain insights into their function and their implications for health and disease. Copyright

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium

SummaryA heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, and approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

Solution structure and dynamics of the outer membrane enzyme PagP by NMR

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-β-d-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel β-barrel preceded by an amphipathic α helix. The β-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual β-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.

Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

Summary Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.

Autoinhibition of the HECT-Type Ubiquitin Ligase Smurf2 through Its C2 Domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

Solution structure of a Nedd4 WW domain-ENaC peptide complex.

Nedd4 is a ubiquitin protein ligase composed of a C2 domain, three (or four) WW domains and a ubiquitin ligase Hect domain. Nedd4 was demonstrated to bind the epithelial sodium channel (αβγENaC), by association of its WW domains with PY motifs (XPPXY) present in each ENaC subunit, and to regulate the cell surface stability of the channel. The PY motif of βENaC is deleted or mutated in Liddle syndrome, a hereditary form of hypertension caused by elevated ENaC activity. Here we report the solution structure of the third WW domain of Nedd4 complexed to the PY motif-containing region of βENaC (TLPIPGTPPPNYDSL, referred to as βP2). A polyproline type II helical conformation is adopted by the PPPN sequence. Unexpectedly, the C-terminal sequence YDSL forms a helical turn and both the tyrosine and the C-terminal leucine contact the WW domain. This is unlike other proline-rich peptides complexed to WW domains, which bind in an extended conformation and lack molecular interactions with residues C-terminal to the tyrosine or the structurally equivalent residue in non-PY motif WW domain targets. The Nedd4 WW domain–ENaC βP2 peptide structure expands our understanding of the mechanisms involved in WW domain–ligand recognition and the molecular basis of Liddle syndrome.

Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase

Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.