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.     

Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains

Src homology 3 (SH3) and WW domains are known to associate with proline-rich motifs within their respective ligands. Here we demonstrate that the proposed adapter protein for Src kinases, Sam68, is a ligand whose proline-rich motifs interact with the SH3 domains of p59(fyn) and phospholipase Cgamma-1 as well as with the WW domains of FBP30 and FBP21. These proline-rich motifs, in turn, are flanked by RG repeats that represent targets for the type I protein arginine N-methyltransferase. The asymmetrical dimethylation of arginine residues within these RG repeats dramatically reduces the binding of the SH3 domains of p59(fyn) and phospholipase Cgamma-1, but has no effect on their binding to the WW domain of FBP30. These results suggest that protein arginine methylation can selectively modulate certain protein-protein interactions and that mechanisms exist for the irreversible regulation of SH3 domain-mediated interactions.     

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.     

Structural characterization of a new binding motif and a novel binding mode in group 2 WW domains

Formin homology 1 (FH1), is a long proline-rich region of formins, shown to bind to five WW containing proteins named formin binding proteins (FBPs). FH1 has several potential binding regions but only the PPLPx motif and its interaction with FBP11WW1 has been characterized structurally. To detect whether additional motifs exist in FH1, we synthesized five peptides and investigated their interaction with FBP28WW2, FBP11WW1 and FBP11WW2 domains. Peptides of sequence PTPPPLPP (positive control), PPPLIPPPP and PPLIPPPP (new motifs) interact with the domains with micromolar affinity. We observed that FBP28WW2 and FBP11WW2 behave differently from FBP11WW1 in terms of motif selection and affinity, since they prefer a doubly interrupted proline stretch of sequence PPLIPP. We determined the NMR structure of three complexes involving the FBP28WW2 domain and the three ligands. Depending on the peptide under study, the domain interacts with two proline residues accommodated in either the XP or the XP2 groove. This difference represents a one-turn displacement of the domain along the ligand sequence. To understand what drives this behavior, we performed further structural studies with the FBP11WW1 and a mutant of FBP28WW2 mimicking the XP2 groove of FBP11WW1. Our observations suggest that the nature of the XP2 groove and the balance of flexibility/rigidity around loop 1 of the domain contribute to the selection of the final ligand positioning in fully independent domains. Additionally, we analyzed the binding of a double WW domain region, FBP11WW1-2, to a long stretch of FH1 using fluorescence spectroscopy and NMR titrations. With this we show that the presence of two consecutive WW domains may also influence the selection of the binding mode, particularly if both domains can interact with consecutive motifs in the ligand. Our results represent the first observation of protein-ligand recognition where a pair of WW and two consecutive motifs in a ligand participate simultaneously.     

WW- and SH3-domain interactions with Epstein-Barr virus LMP2A

Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.     

Formin binding proteins bear WWP/WW domains that bind proline-rich peptides and functionally resemble SH3 domains.

The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for Src homology 3 (SH3) ligands. To identify proteins that interact with formins, we used this proline-rich region to screen mouse limb bud expression libraries for formin binding proteins (FBPs). As expected, we found one class of FBPs that contains SH3 domains, including two novel members of this class. In addition, however, we also found a novel class of FBPs that contains one or two copies of a 26 amino acid homology region that has been recently termed the WWP or WW motif. We demonstrate that WWP/WW domains as short as 26 amino acids can act as modular protein-binding interfaces that bind with high affinity to proline-rich sequences that are similar and, in some cases, identical to SH3 ligands. Furthermore, we find that the WWP/WW domain can compete with the Abl SH3 domain in binding a proline-rich peptide present in formin. Our results suggest that these novel protein interaction domains can perform functions similar to those of SH3 domains and, thus, might regulate SH3 interactions with target proteins through competitive binding.     

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.     

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.     

Interaction between the N-terminal SH3 domain of Nck-alpha and CD3-epsilon-derived peptides: non-canonical and canonical recognition motifs

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.     

Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.

Short contiguous peptides harboring proline-rich motifs are frequently involved in protein-protein interactions, such as associations with Src homology 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are distinct, suggesting that additional protein families exist that use stacked aromatic amino acids (AA domains) to bind polyproline motifs [1] [2] [3]. A polyproline motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytogenes, serves as a ligand for the Ena/VASP protein family --the vasodilator-stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabled (Ena) and the Ena/VASP-like protein Evl [4] [5] [6] [7]. These share a similar overall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5]. Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residues within two conserved hydrophilic regions of the EVH1 domain abolished binding to ActA. Binding of an EVH1 mutant with reversed charges could partially be rescued by introducing complementary mutations within the ligand. Like SH3 domains, aromatic residues within the EVH1 domain interacted with polyprolines, whereas the ligand specificity of either domain was determined by reciprocally charged residues. The EVH1 domain is therefore a new addition to the AA domain superfamily, which includes SH3 and WW domains.     

Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains

To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.     

Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

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.     

Defining the specificity space of the human SRC homology 2 domain

Src homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanating from protein-tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide binding properties of 76 SH2 domains by screening an oriented peptide array library. Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. We identified a number of novel binding motifs, which are exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P + 4 relative to the Tyr(P) residue. Based on the oriented peptide array library data, we developed scoring matrix-assisted ligand identification (or SMALI), a Web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP (SLAM-associated protein), a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells.     

A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.     

Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function.

The Ena-VASP homology (EVH1) domain is a protein interaction module found in several proteins that are involved in transducing migratory and morphological signals into cytoskeletal reorganization. EVH1 specifically recognizes proline-rich sequences in its binding partners and directs the localization and formation of multicomponent assemblies involved in actin-based motile processes and neural development. The structure of the complex between an EVH1 domain and the target peptide sequence EFPPPPT identifies the interactions responsible for recognition and distinguishes it from other proline-rich binding modules, including SH3 and WW domains. Surprisingly, the EVH1 domain has structural similarity to pleckstrin homology (PH), phosphotyrosine-binding (PTB) and ran-binding (RanBD) domains.     

Structural basis for specific binding of the Gads SH3 domain to an RxxK motif-containing SLP-76 peptide: a novel mode of peptide recognition

The SH3 domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 domain and upon binding adopts a unique structure characterized by a right-handed 3(10) helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 domains.     

Structure of FBP11 WW1-PL ligand complex reveals the mechanism of proline-rich ligand recognition by group II/III WW domains

FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.     

A dynamic view of domain-motif interactions

Many protein-protein interactions are mediated by domain-motif interaction, where a domain in one protein binds a short linear motif in its interacting partner. Such interactions are often involved in key cellular processes, necessitating their tight regulation. A common strategy of the cell to control protein function and interaction is by post-translational modifications of specific residues, especially phosphorylation. Indeed, there are motifs, such as SH2-binding motifs, in which motif phosphorylation is required for the domain-motif interaction. On the contrary, there are other examples where motif phosphorylation prevents the domain-motif interaction. Here we present a large-scale integrative analysis of experimental human data of domain-motif interactions and phosphorylation events, demonstrating an intriguing coupling between the two. We report such coupling for SH3, PDZ, SH2 and WW domains, where residue phosphorylation within or next to the motif is implied to be associated with switching on or off domain binding. For domains that require motif phosphorylation for binding, such as SH2 domains, we found coupled phosphorylation events other than the ones required for domain binding. Furthermore, we show that phosphorylation might function as a double switch, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. Evolutionary analysis shows that co-evolution of the motif and the proximal residues capable of phosphorylation predominates over other evolutionary scenarios, in which the motif appeared before the potentially phosphorylated residue, or vice versa. Our findings provide strengthening evidence for coupled interaction-regulation units, defined by a domain-binding motif and a phosphorylated residue.