Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.     

Can we infer peptide recognition specificity mediated by SH3 domains?

Protein interaction domain families that modulate the formation of macromolecular complexes recognize specific sequence or structural motifs. For instance SH3 and WW domains bind to polyproline peptides while SH2 and FHA domains bind to peptides phosphorylated in Tyr and Thr respectively. Within each family, variations in the chemical characteristics of the domain binding pocket modulate a finer peptide recognition specificity and, as a consequence, determine the selection of functional protein partners in vivo. In the proteomic era there is the need for reliable inference methods to help restricting the sequence space of the putative targets to be confirmed experimentally by more laborious experimental approaches. Here we will review the published data about the peptide recognition specificity of the SH3 domain family and we will propose a classification of SH3 domains into eight classes. Finally, we will discuss whether the available information is sufficient to infer the recognition specificity of any uncharacterized SH3 domain.     

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.     

Promiscuous binding nature of SH3 domains to their target proteins

SH3 domains are small but important domains in cell-signaling and function through protein-protein interactions. Their promiscuous nature in binding to polyproline peptides makes them much more important because many SH3 domains from different proteins bind to different proteins having polyproline template on their surface. Very subtle changes in the sequence of SH3 domains and the binding peptides determine the specificity of the peptide binding. Recent observation that SH3 domains bind to non- proline peptides makes the scenario of peptide binding involving SH3 domains complicated. If domain swapped dimerization as observed in Eps8-SH3 domain also binds different peptides, it proves the versatility of the SH3 domains in binding to peptides in various ways. An overview of the promiscuity of SH3 domains has been discussed.     

Structural basis of PxxDY motif recognition in SH3 binding

We have determined the solution structure of epidermal growth factor receptor pathway substrate 8 (Eps8) L1 Src homology 3 (SH3) domain in complex with the PPVPNPDYEPIR peptide from the CD3ε cytoplasmic tail. Our structure reveals the distinct structural features that account for the unusual specificity of the Eps8 family SH3 domains for ligands containing a PxxDY motif instead of canonical PxxP ligands. The CD3ε peptide binds Eps8L1 SH3 in a class II orientation, but neither adopts a polyproline II helical conformation nor engages the first proline-binding pocket of the SH3 ligand binding interface. Ile531 of Eps8L1 SH3, instead of Tyr or Phe residues typically found in this position in SH3 domains, renders this hydrophobic pocket smaller and nonoptimal for binding to conventional PxxP peptides. A positively charged arginine at position 512 in the n-Src loop of Eps8L1 SH3 plays a key role in PxxDY motif recognition by forming a salt bridge to D7 of the CD3ε peptide. In addition, our structural model suggests a hydrogen bond between the hydroxyl group of the aromatic ring of Y8 and the carboxyl group of E496, thus explaining the critical role of the PxxDY motif tyrosine residue in binding to Eps8 family SH3. These finding have direct implications also for understanding the atypical binding specificity of the amino-terminal SH3 of the Nck family proteins.     

Systematic discovery of new recognition peptides mediating protein interaction networks

Many aspects of cell signalling, trafficking, and targeting are governed by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or “linear motif” (e.g., SH3 binding to PxxP). Many domains are known, though comparatively few linear motifs have been discovered. Their short length (three to eight residues), and the fact that they often reside in disordered regions in proteins makes them difficult to detect through sequence comparison or experiment. Nevertheless, each new motif provides critical molecular details of how interaction networks are constructed, and can explain how one protein is able to bind to very different partners. Here we show that binding motifs can be detected using data from genome-scale interaction studies, and thus avoid the normally slow discovery process. Our approach based on motif over-representation in non-homologous sequences, rediscovers known motifs and predicts dozens of others. Direct binding experiments reveal that two predicted motifs are indeed protein-binding modules: a DxxDxxxD protein phosphatase 1 binding motif with a K_D of 22 μM and a VxxxRxYS motif that binds Translin with a K_D of 43 μM. We estimate that there are dozens or even hundreds of linear motifs yet to be discovered that will give molecular insight into protein networks and greatly illuminate cellular processes.     

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.     

Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain

Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3) domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders) and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.     

The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation

Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly. The C terminus of nebulin, located in the sarcomere Z-disk, comprises an SH3 domain, a module well known for its role in protein/protein interactions. SH3 domains are known to recognize proline-rich ligands, which have been classified as type I or type II, depending on their relative orientation with respect to the SH3 domain in the complex formed. Type I ligands are bound with their N terminus at the RT loop of the SH3 domain, while type II ligands are bound with their C terminus at the RT loop. Many SH3 domains can bind peptides of either class. Despite the potential importance of the SH3 domain for the function of nebulin as an integral part of a complex network of interactions, no in vivo partner has been identified so far. We have adopted an integrated approach, which combines bioinformatic tools with experimental validation to identify possible partners of nebulin SH3. Using the program SPOT, we performed an exhaustive screening of the muscle sequence databases. This search identified a number of potential nebulin SH3 partners, which were then tested experimentally for their binding affinity. Synthetic peptides were studied by both fluorescence and NMR spectroscopy. Our results show that nebulin SH3 domain binds selectively to type II peptides. The affinity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 microM). This affinity is among the highest found for SH3/peptide complexes, suggesting that the identified stretch could have significance in vivo. The strategy outlined here is of more general applicability and may provide a valuable tool to identify potential partners of SH3 domains and of other peptide-binding modules.     

Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.     

A novel pro-Arg motif recognized by WW domains

WW domains mediate protein-protein interactions through binding to short proline-rich sequences. Two distinct sequence motifs, PPXY and PPLP, are recognized by different classes of WW domains, and another class binds to phospho-Ser-Pro sequences. We now describe a novel Pro-Arg sequence motif recognized by a different class of WW domains using data from oriented peptide library screening, expression cloning, and in vitro binding experiments. The prototype member of this group is the WW domain of formin-binding protein 30 (FBP30), a p53-regulated molecule whose WW domains bind to Pro-Arg-rich cellular proteins. This new Pro-Arg sequence motif re-classifies the organization of WW domains based on ligand specificity, and the Pro-Arg class now includes the WW domains of FBP21 and FE65. A structural model is presented which rationalizes the distinct motifs selected by the WW domains of YAP, Pin1, and FBP30. The Pro-Arg motif identified for WW domains often overlaps with SH3 domain motifs within protein sequences, suggesting that the same extended proline-rich sequence could form discrete SH3 or WW domain complexes to transduce distinct cellular signals.     

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.     

Ubiquitin binds to and regulates a subset of SH3 domains

SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that bind to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain and that ubiquitin binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Lipid-binding hSH3 domains in immune cell adapter proteins

SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich sequences within intracellular proteins. More recently, binding of SH3 domains to unusual peptide motifs, folded proteins or lipids has been reported. Here we show that the newly defined hSH3 domains of immune cell adapter proteins bind lipid membranes with distinct affinities. The interaction of the hSH3 domains of adhesion and degranulation promoting adapter protein (ADAP) and PRAM-1 (Promyelocytic-Retinoic acid receptor alpha target gene encoding an Adaptor Molecule-1), with phosphatidylcholine-containing liposomes is observed upon incorporation of phosphatidylserine (PS) or phosphoinositides (PIs) into the membrane bilayer. Mechanistically we show that stable association of the N-terminal, amphipathic helix with the beta-sheet scaffold favours lipid binding and that the interaction with PI(4,5)P(2)-containing liposomes is consistent with a single-site, non-cooperative binding mechanism. Functional investigations indicate that deletion of both amphipathic helices of the hSH3 domains reduces the ability of ADAP to enhance adhesion and migration in stimulated T cells.     

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (approximately 31 and 3.4 microM, respectively), whereas the other four peptides bind weakly (>100 microM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly.     

The SH3 domain of a M7 interacts with its C-terminal proline-rich region

Myosins play essential roles in migration, cytokinesis, endocytosis, and adhesion. They are composed of a large N-terminal motor domain with ATPase and actin binding sites and C-terminal neck and tail regions, whose functional roles and structural context in the protein are less well characterized. The tail regions of myosins I, IV, VII, XII, and XV each contain a putative SH3 domain that may be involved in protein-protein interactions. SH3 domains are reported to bind proline-rich motifs, especially "PxxP" sequences, and such interactions serve regulatory functions. The activity of Src, PI3, and Itk kinases, for example, is regulated by intramolecular interactions between their SH3 domain and internal proline-rich sequences. Here, we use NMR spectroscopy to reveal the structure of a protein construct from Dictyostelium myosin VII (DdM7) spanning A1620-T1706, which contains its SH3 domain and adjacent proline-rich region. The SH3 domain forms the signature beta-barrel architecture found in other SH3 domains, with conserved tryptophan and tyrosine residues forming a hydrophobic pocket known to bind "PxxP" motifs. In addition, acidic residues in the RT or n-Src loops are available to interact with the basic anchoring residues that are typically found in ligands or proteins that bind SH3 domains. The DdM7 SH3 differs in the hydrophobicity of the second pocket formed by the 3(10) helix and following beta-strand, which contains polar rather than hydrophobic side chains. Most unusual, however, is that this domain binds its adjacent proline-rich region at a surface remote from the region previously identified to bind "PxxP" motifs. The interaction may affect the orientation of the tail without sacrificing the availability of the canonical "PxxP"-binding surface.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A beta-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.