An autonomously folding beta-hairpin derived from the human YAP65 WW domain: attempts to define a minimum ligand-binding motif

WW domains are broadly distributed among natural proteins; these modules play a role in bringing specific proteins together. The ligands recognized by WW domains are short segments rich in proline residues. We have tried to identify the minimum substructure within a WW domain that is required for ligand binding. WW domains typically comprise ca. 40 residues and fold to a three-stranded beta-sheet. Structural data for several WW domain/ligand complexes suggest that most or all of the intermolecular contacts involve beta-strands 2 and 3. We have developed a 16-residue peptide that folds to a beta-hairpin conformation that appears to mimic beta-strands 2 and 3 of the human YAP65 WW domain, but this peptide does not bind to known ligands. Thus, the minimum binding domain is larger than the latter two strands of the WW domain beta-sheet.     

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.     

Structural analysis of WW domains and design of a WW prototype

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.     

Design and NMR conformational study of a beta-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein beta-sheets is de novo protein design. To obtain a three-stranded beta-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed beta-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural beta-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt beta-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest beta-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel beta-sheet structure with a disordered C-terminal tail. To discern between the contributions to beta-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. beta-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to beta-sheet stability were drawn by comparing structural properties of these four peptides.     

WW and SH3 domains, two different scaffolds to recognize proline-rich ligands

WW domains are small protein modules composed of approximately 40 amino acids. These domains fold as a stable, triple stranded beta-sheet and recognize proline-containing ligands. WW domains are found in many different signaling and structural proteins, often localized in the cytoplasm as well as in the cell nucleus. Based on analyses of seven structures of WW domains, we discuss their diverse binding preferences and sequence conservation patterns. While modeling WW domains for which structures have not been determined we uncovered a case of potential molecular and functional convergence between WW and SH3 domains. The binding surface of the modeled WW domain of Npw38 protein shows a remarkable similarity to the SH3 domain of Sem5 protein, confirming biochemical data on similar binding predilections of both domains.     

Stability of the beta-sheet of the WW domain: A molecular dynamics simulation study.

The WW domain consists of approximately 40 residues, has no disulfide bridges, and forms a three-stranded antiparallel beta-sheet that is monomeric in solution. It thus provides a model system for studying beta-sheet stability in native proteins. We performed molecular dynamics simulations of two WW domains, YAP65 and FBP28, with very different stability characteristics, in order to explore the initial unfolding of the beta-sheet. The less stable YAP domain is much more sensitive to simulation conditions than the FBP domain. Under standard simulation conditions in water (with or without charge-balancing counterions) at 300 K, the beta-sheet of the YAP WW domain disintegrated at early stages of the simulations. Disintegration commenced with the breakage of a hydrogen bond between the second and third strands of the beta-sheet due to an anticorrelated transition of the Tyr-28 psi and Phe-29 phi angles. Electrostatic interactions play a role in this event, and the YAP WW domain structure is more stable when simulated with a complete explicit model of the surrounding ionic strength. Other factors affecting stability of the beta-sheet are side-chain packing, the conformational entropy of the flexible chain termini, and the binding of cognate peptide.     

Characterization of the structure and function of W --> F WW domain variants: identification of a natively unfolded protein that folds upon ligand binding

The WW domain adopts a compact, three-stranded, antiparallel beta-sheet structure that mediates protein-protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity and for ligand binding could be evaluated. A biophysical approach was employed to compare the W17F, W39F, and W17F/W39F WW domains to the wild-type protein. The data demonstrate that replacement of Trp39 with Phe (W39F) does not disrupt the structure of the WW domain variant, but does abolish ligand binding. In contrast, the W17F WW domain variant is largely if not completely unfolded; however, this variant undergoes a PY-ligand induced disorder to order (folding) transition. The dissociation constant for the W17F WW domain-PY-ligand interaction is 15.1 +/- 1.2 microM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 +/- 0.33 microM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.     

A map of WW domain family interactions

WW domains are protein modules that bind proline-rich ligands. WW domain-ligand complexes are of importance as they have been implicated in several human diseases such as muscular dystrophy, cancer, hypertension, Alzheimer's, and Huntington's diseases. We report the results of a protein array aimed at mapping all the human WW domain protein-protein interactions. Our biochemical approach integrates parallel synthesis of peptides, protein expression, and high-throughput screening methodology combined with tools of bioinformatics. The results suggest that the majority of the bioinformatically predicted WW peptide ligands and most WW domains are functional, and that only about 10% of the measured domain-ligand interactions are positive. The analysis of the WW domain protein arrays also underscores the importance of the amino acid residues surrounding the WW ligand core motifs for specific binding to WW domains. In addition, the methodology presented here allows for the rapid elucidation of WW domain-ligand interactions with multiple applications including prediction of exact WW ligand binding sites, which can be applied to the mapping of other protein signaling domain families. Such information can be applied to the generation of protein interaction networks and identification of potential drug targets. To our knowledge, this report describes the first protein-protein interaction map of a domain in the human proteome.     

NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray crystal structures of Pin1.

The NMR solution structure of the isolated Apo Pin1 WW domain (6-39) reveals that it adopts a twisted three-stranded antiparallel beta-sheet conformation, very similar to the structure exhibited by the crystal of this domain in the context of the two domain Pin1 protein. While the B factors in the apo x-ray crystal structure indicate that loop 1 and loop 2 are conformationally well defined, the solution NMR data suggest that loop 1 is quite flexible, at least in the absence of the ligand. The NMR chemical shift and nuclear Overhauser effect pattern exhibited by the 6-39 Pin1 WW domain has proven to be diagnostic for demonstrating that single site variants of this domain adopt a normally folded structure. Knowledge of this type is critical before embarking on time-consuming kinetic and thermodynamic studies required for a detailed understanding of beta-sheet folding.     

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.     

Understanding the mechanism of beta-sheet folding from a chemical and biological perspective

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.     

Beta-sheet folding mechanisms from perturbation energetics

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.     

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.     

The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.     

Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding

An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.     

Solution structure and binding specificity of FBP11/HYPA WW domain as Group-II/III

The Group-II/III WW domains bind Pro-rich sequences, the most frequent protein motif found in eucaryotic genomes. We have proposed that the Group-II and -III WW domains be merged into a larger group because the members of each group have relatively wide specificity and bind to the common ligands [Kato et al., J Biol Chem 2004;279:31833-31841]. We have also proposed that Group-II/III has a common surface patch, the XP2 groove, to bind the ligands. The first WW domain of FBP11/HYPA is one of the Group-II/III WW domains. The solution structure of the 26 residue-long converged region exhibits an antiparallel triple stranded beta-sheet with a small hydrophobic core. The WW domain of FBP11/HYPA has both XP and XP2 grooves on its surface. Ligand titration by 1H-15N HSQC NMR spectra revealed that the WW domain of FBP11/HYPA binds all the peptides with the PL, PP, and PR motifs. The profile patterns of chemical shift perturbation were quite similar among the spectra titrated with all three ligands. In addition, the titration significantly shifts the signals of the residues that compose the XP2 groove. All these findings suggest the functional importance of the XP2 groove and group definition of Group-II/III of the WW domains.     

Comparative structural and energetic analysis of WW domain-peptide interactions

WW domains are small globular protein interaction modules found in a wide spectrum of proteins. They recognize their target proteins by binding specifically to short linear peptide motifs that are often proline-rich. To infer the determinants of the ligand binding propensities of WW domains, we analyzed 42 WW domains. We built models of the 3D structures of the WW domains and their peptide complexes by comparative modeling supplemented with experimental data from peptide library screens. The models provide new insights into the orientation and position of the peptide in structures of WW domain-peptide complexes that have not yet been determined experimentally. From a protein interaction property similarity analysis (PIPSA) of the WW domain structures, we show that electrostatic potential is a distinguishing feature of WW domains and we propose a structure-based classification of WW domains that expands the existent ligand-based classification scheme. Application of the comparative molecular field analysis (CoMFA), GRID/GOLPE and comparative binding energy (COMBINE) analysis methods permitted the derivation of quantitative structure-activity relationships (QSARs) that aid in identifying the specificity-determining residues within WW domains and their ligand-recognition motifs. Using these QSARs, a new group-specific sequence feature of WW domains that target arginine-containing peptides was identified. Finally, the QSAR models were applied to the design of a peptide to bind with greater affinity than the known binding peptide sequences of the yRSP5-1 WW domain. The prediction was verified experimentally, providing validation of the QSAR models and demonstrating the possibility of rationally improving peptide affinity for WW domains. The QSAR models may also be applied to the prediction of the specificity of WW domains with uncharacterized ligand-binding properties.     

A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.

WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (betaB5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (betaB7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second beta-sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.     

Structural characterisation of PinA WW domain and a comparison with other group IV WW domains, Pin1 and Ess1

The NMR solution structure of the PinA WW domain from Aspergillus nidulans is presented. The backbone of the PinA WW domain is composed of a triple-stranded anti-parallel beta-sheet and an alpha-helix similar to Ess1 and Pin1 without the alpha-helix linker. Large RMS deviations in Loop I were observed both from the NMR structures and molecular dynamics simulation suggest that the Loop I of PinA WW domain is flexible and solvent accessible, thus enabling it to bind the pS/pT-P motif. The WW domain in this structure are stabilised by a hydrophobic core. It is shown that the linker flexibility of PinA is restricted because of an alpha-helical structure in the linker region. The combination of NMR structural data and detailed Molecular Dynamics simulations enables a comprehensive structural and dynamic understanding of this protein.