SMART MODE:

NORMAL GENOMIC

• Sudol M, Sliwa K, Russo T
• Functions of WW domains in the nucleus.
• FEBS Lett. 2001; 490: 190-5
• Display abstract

• The WW domain is a protein module found in a wide range of signaling proteins. It is one of the smallest protein modules that folds as a monomer without disulfide bridges or cofactors. WW domains bind proteins containing short linear peptide motifs that are proline-rich or contain at least one proline. Although the WW domain was initially considered a 'cytoplasmic module', the proteins containing WW domains have also been localized in the cell nucleus. Moreover, these proteins have been documented to participate in co-activation of transcription and modulation of RNA polymerase II activity. The carboxy-terminal domain (CTD) of RNA polymerase II acts as an assembly platform for distinct WW domain-containing proteins that affect the function of the RNA polymerase II. The formation of complexes between CTD and WW domain-containing proteins is regulated by phosphorylation of the CTD. Since the CTD sequence is highly repetitive and a target of several post-translational modifications and conformational changes, it presents a unique structure capable of enormous molecular diversity. The WW domain has been implicated in several human diseases including Alzheimer's disease. The WW domain-containing iso-prolyl isomerase named Pin1, a protein known to be essential for cell cycle progression, was shown to be active in restoration of the microtubule-binding activity of Tau, a protein of neurofibrillar tangles found in the brains of Alzheimer's patients. It is the WW domain of Pin1 that interacts directly with Tau protein. In addition, the WW domain-containing adapter protein FE65 was shown to regulate processing of Alzheimer's amyloid precursor protein. It is expected that by understanding the details of the WW domain-mediated protein-protein interactions, we will be able to illuminate numerous signaling pathways which control certain aspects of transcription and cell cycle.

• Rousseau F, Schymkowitz JW, Wilkinson HR, Itzhaki LS
• Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues.
• Proc Natl Acad Sci U S A. 2001; 98: 5596-601
• Display abstract

• p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer-dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

• Guillebault D, Derelle E, Bhaud Y, Moreau H
• Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription.
• Protist. 2001; 152: 127-38
• Display abstract

• Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.

• Toepert F, Pires JR, Landgraf C, Oschkinat H, Schneider-Mergener J
• Cover Picture.
• Angew Chem Int Ed Engl. 2001; 40: 805-805
• Display abstract

• The cover picture shows an array of several hundred synthetically produced variants of the 44 amino acids comprising the hyAP-WW protein domain. The array was produced by a stepwise SPOT synthesis on a cellulose membrane. At each synthesis site (spot) a WW domain is bound to the membrane through a C-terminal ester bond. The secondary structure of the WW domain is shown in green as a ribbon. The domains of the single spots differ only by a single amino acid. All the domains were tested simultaneously for their ability to bind to a peptide motif (red) common to many proteins. The binding was evident when visualized by chemoluminescence, with the Spots having various intensities. The systematic analysis undertaken here enabled molecular biology to be carried out that would have otherwise have required great effort, or not been done at all. This chemical technique also allows the construction of many non-genetically coded building blocks. Through the use of modern synthetic techniques for the coupling of peptides this method should also allow the synthesis of proteins. The combination of molecular biological and chemical methods opens up opportunities for the preparation of protein chips for diagnostics and drug discovery. More about this can be found in the article by Schneider-Mergener et al. on p. 897ff.

• ter Haar E, Harrison SC, Kirchhausen T
• Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin.
• Proc Natl Acad Sci U S A. 2000; 97: 1096-100
• Display abstract

• The "WD40" domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a beta-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, beta-arrestin 2 and the beta-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar "clathrin-box" motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which beta-propellers recognize specific target proteins.

• Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, Richard S
• Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains.
• J Biol Chem. 2000; 275: 16030-6
• Display abstract

• 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.

• Matsuda S et al.
• Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3.
• Biochim Biophys Acta. 2000; 1491: 321-6
• Display abstract

• A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.

• Kanelis V, Donaldson L, Muhandiram DR, Rotin D, Forman-Kay JD, Kay LE
• Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes.
• J Biomol NMR. 2000; 16: 253-9
• Display abstract

• Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even polyproline stretches. The lack of amide protons in such regions complicates assignment, since 1HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the protein Crk with a region containing 9 prolines in a 14 amino acid sequence, as well as a WW domain that interacts with a proline-rich target. A modified version of the HACAN pulse scheme, originally described by Bax and co-workers [Wang et al. (1995) J. Biomol. NMR, 5, 376-382], and an experiment which correlates the intra-residue 1Halpha, 13Calpha/13Cbeta chemical shifts with the 15N shift of the subsequent residue are presented and applied to the two systems listed above, allowing sequential assignment of the molecules.

• Zarrinpar A, Lim WA
• Converging on proline: the mechanism of WW domain peptide recognition.
• Nat Struct Biol. 2000; 7: 611-3

• Liu Z et al.
• Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain.
• Nature. 2000; 408: 1004-8
• Display abstract

• The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.

• Huang X, Poy F, Zhang R, Joachimiak A, Sudol M, Eck MJ
• Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan.
• Nat Struct Biol. 2000; 7: 634-8
• Display abstract

• Dystrophin and beta-dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of beta-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of beta-dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in beta-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The beta-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.

• Greener MJ, Roberts RG
• Conservation of components of the dystrophin complex in Drosophila.
• FEBS Lett. 2000; 482: 13-8
• Display abstract

• Defects in the dystrophin complex (DC) underlie several human genetic disorders, but our dissection of its function is complicated by potential redundancy of the multiple vertebrate isoforms of most DC components. We here complete our previous description of Drosophila dystrophin, and show that the fly retains all essential components of the DC, but with substantially less diversity. Seventeen known human components (three dystrophin-related proteins, two dystrobrevins, five sarcoglycans, five syntrophins, one dystroglycan and one sarcospan) appear to be reduced to eight in Drosophila (one, one, three, two, one and none, respectively). The simplicity of this system recommends it as a model for its human counterpart.

• Waragai M et al.
• PQBP-1/Npw38, a nuclear protein binding to the polyglutamine tract, interacts with U5-15kD/dim1p via the carboxyl-terminal domain.
• Biochem Biophys Res Commun. 2000; 273: 592-5
• Display abstract

• PQBP-1 was identified as a binding protein to the polyglutamine tract present in various transcription-related factors and causative genes for neurodegenerative disorders. This novel gene contains at least two functional domains, WW domain and carboxyl-terminal domain (CTD), strictly conserved beyond species. Although human PQBP-1 additionally contains the polar amino acid-rich domain by which it binds to the polyglutamine tract, genuine physiological function(s) have not been clarified. In this study, we showed that U5-15kD, human homologue of fission yeast dim1p, is a partner molecule of PQBP-1 binding to CTD. This finding suggests physiological functions of PQBP-1 in splicing, cell cycle, and ubiquitination, through which we can speculate the pathological roles of PQBP-1 in triplet repeat diseases.

• Dalby PA, Hoess RH, DeGrado WF
• Evolution of binding affinity in a WW domain probed by phage display.
• Protein Sci. 2000; 9: 2366-76
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• The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.

• Cestra G et al.
• The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.
• J Biol Chem. 1999; 274: 32001-7
• Display abstract

• The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

• Komuro A, Saeki M, Kato S
• Association of two nuclear proteins, Npw38 and NpwBP, via the interaction between the WW domain and a novel proline-rich motif containing glycine and arginine.
• J Biol Chem. 1999; 274: 36513-9
• Display abstract

• We have previously reported a nuclear protein possessing a WW domain, Npw38 (Komuro, A., Saeki, M., and Kato, S. (1999) Nucleic Acids Res. 27, 1957-1965). Here we report a Npw38-binding protein, NpwBP, isolated from HeLa cell nuclear extracts and its characterization using a cloned cDNA. NpwBP contains two proline-rich regions that are capable of binding to the WW domain of Npw38. The binding analysis using an oligopeptide-immobilized membrane revealed that the WW domain of Npw38 preferentially recognizes a short proline-rich sequence, PPGPPP, surrounded by an arginine residue, so we named it a PGR motif. Localization analysis using green fluorescent protein fusion protein and immunostaining showed that Npw38 and NpwBP are colocalized in the same subnuclear region. Coimmunoprecipitation experiments confirmed the association between Npw38 and NpwBP, which were expressed as epitope-tagged forms in COS7 cells. Furthermore, the N-terminal region of NpwBP has binding ability for poly(rG) and G-rich single-stranded DNA. These results suggest that NpwBP is a physiological ligand of Npw38 and that the Npw38-NpwBP complex may function as a component of an mRNA factory in the nucleus.

• Mahoney NM, Rozwarski DA, Fedorov E, Fedorov AA, Almo SC
• Profilin binds proline-rich ligands in two distinct amide backbone orientations.
• Nat Struct Biol. 1999; 6: 666-71
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• 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.

• Golding GB
• Simple sequence is abundant in eukaryotic proteins.
• Protein Sci. 1999; 8: 1358-61
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• All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins. Proteins that are recently evolutionarily related were excluded. The most frequently present protein segments are long, tandem repetitions of a single amino acid. For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them. These peptide segments may not be functional protein domains. Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs.

• Espanel X, Sudol M
• A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.
• J Biol Chem. 1999; 274: 17284-9
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• 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.

• Koepf EK, Petrassi HM, Ratnaswamy G, Huff ME, Sudol M, Kelly JW
• Characterization of the structure and function of W --> F WW domain variants: identification of a natively unfolded protein that folds upon ligand binding.
• Biochemistry. 1999; 38: 14338-51
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• 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.

• Nguyen JT, Turck CW, Cohen FE, Zuckermann RN, Lim WA
• Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors.
• Science. 1998; 282: 2088-92
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• Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.

• Dho SE, Jacob S, Wolting CD, French MB, Rohrschneider LR, McGlade CJ
• The mammalian numb phosphotyrosine-binding domain. Characterization of binding specificity and identification of a novel PDZ domain-containing numb binding protein, LNX.
• J Biol Chem. 1998; 273: 9179-87
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• Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during development. A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for Numb function. Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain. Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and 70 kDa (LNX-b). LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains. The unique amino-terminal region of LNX includes a RING finger domain. The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY, found in both protein isoforms. Mutational analysis of LNX and peptide competition experiments showed that phosphorylation of the tyrosine residue within this motif was not required for binding to the Numb PTB domain. Finally, we also provide evidence that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent binding of other PTB domain-containing proteins such as SHC. We speculate that LNX may be important for clustering PTB-containing proteins with functionally related transmembrane proteins in specific membrane compartments.

• Masuda ES, Huang BC, Fisher JM, Luo Y, Scheller RH
• Tomosyn binds t-SNARE proteins via a VAMP-like coiled coil.
• Neuron. 1998; 21: 479-80

• Grabs D et al.
• The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence.
• J Biol Chem. 1997; 272: 13419-25
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• Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.

• Rajan R, Awasthi SK, Bhattacharjya S, Balaram P
• "Teflon-coated peptides": hexafluoroacetone trihydrate as a structure stabilizer for peptides.
• Biopolymers. 1997; 42: 125-8

• Linn H, Ermekova KS, Rentschler S, Sparks AB, Kay BK, Sudol M
• Using molecular repertoires to identify high-affinity peptide ligands of the WW domain of human and mouse YAP.
• Biol Chem. 1997; 378: 531-7
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• The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.

• Sugiyama S, Igarashi K, Morikawa K
• [PotD protein: three-dimensional structure and function]
• Seikagaku. 1997; 69: 113-9

• Sadoulet-Puccio HM, Rajala M, Kunkel LM
• Dystrobrevin and dystrophin: an interaction through coiled-coil motifs.
• Proc Natl Acad Sci U S A. 1997; 94: 12413-8
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• Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins' homologous coiled-coil motifs that flank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.

• Shpakov AO, Derkach KV
• [Comparative analysis of primary structures of yeast dolicholphosphomannosyl- and dolichophosphoglucosyl synthetases and other dolichol-conjugated enzymes]
• Ukr Biokhim Zh. 1996; 68: 64-75
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• Primary structure of yeast dolicholphosphomannosyl- (DPMS) and dolicholphosphoglucosylsynthetases (DPGS) have been compared both between themselves and DPMS and DPGS with yeast beta- and alpha-1, 3-mannosyltransferases, glucosyltransferases and rat mannosyl-binding proteins. The long homological segments were revealed. The homological segments of beta- and alpha-1, 3-mannosyltransferases were located in regions having potency to form coiled-coil structures. These structures are known to be included in carbohydrate-binding protein domains. The previous data and the results presented now have made it possible to conclude that the dolichol-coupled enzymes have evolutionary relationship between themselves and common evolutionary roots with carbohydrate-binding proteins (lectins).

• Hall MN
• The TOR signalling pathway and growth control in yeast.
• Biochem Soc Trans. 1996; 24: 234-9

• Bork P, Gibson TJ
• Applying motif and profile searches.
• Methods Enzymol. 1996; 266: 162-84

• Bressler SL et al.
• cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein.
• Hum Mol Genet. 1996; 5: 1589-98
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• Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.

• Chan DC, Bedford MT, Leder P
• Formin binding proteins bear WWP/WW domains that bind proline-rich peptides and functionally resemble SH3 domains.
• EMBO J. 1996; 15: 1045-54
• Display abstract

• 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.

• Winder SJ, Gibson TJ, Kendrick-Jones J
• Low probability of dystrophin and utrophin coiled coil regions forming dimers.
• Biochem Soc Trans. 1996; 24: 280-280

• Staub O, Rotin D
• WW domains.
• Structure. 1996; 4: 495-9
• Display abstract

• WW domains are recently described protein-protein interaction modules; they bind to proline-rich sequences that usually also contain a tyrosine. These domains have been detected in several unrelated proteins, often alongside other domains. Recent studies suggest that WW domains in specific proteins may play a role in diseases such as hypertension or muscular dystrophy.

• Einbond A, Sudol M
• Towards prediction of cognate complexes between the WW domain and proline-rich ligands.
• FEBS Lett. 1996; 384: 1-8
• Display abstract

• The WW domain is a structured protein module found in a wide range of regulatory, cytoskeletal, and signaling molecules. Its ligands contain proline-rich sequences, some of which show a core consensus of XPPXY that is critical for binding. In order to gain a better understanding of the molecular and biological functions of WW domains, we decided to predict their cognate ligands by searching databases for proteins containing the XPPXY consensus. Using several axioms that take into account evolutionary conservation and functional similarity, we have identified four groups of proteins representing candidate ligands that signal through known or unknown WW domains. These include viral Gag proteins, sodium channels, interleukin receptors, and a subgroup of serine/threonine kinases. In addition, we proposed that dystrophin and beta-dystroglycan bind through the WW-XPPXY link and that interference with this interaction could result in muscular dystrophy. Our study provides guidelines for experiments to probe the molecular and biological functions of the WW domain-ligand connection. Should these predictions be proven empirically, the results may have important ramifications for basic research and medicine.

• Harrison SC
• Peptide-surface association: the case of PDZ and PTB domains.
• Cell. 1996; 86: 341-3

• Wang B et al.
• Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene.
• Oncogene. 1996; 12: 1921-9
• Display abstract

• Arg and c-Abl represent the mammalian members of the Abelson family of nonreceptor protein-tyrosine kinases. To gain insight into the biological role of Arg we used the two-hybrid approach to identify interacting proteins. Using a C-terminal segment of Arg we identified a novel protein, ArgBP1 (Arg binding protein 1). ArgBP1 contains a C-terminal SH3 domain, several PEST sequences, a serine rich domain and an SH3 binding site. ArgBP1 is ubiquitously expressed as two transcripts of approximately 2.2 kb and approximately 8 kb with highest levels in brain, heart and testis. The association of ArgBP1 with Arg in living cells was confirmed by coimmunoprecipitation in cotransfected COS cells. Analysis of the mechanism of association indicated that the ArgBP1 SH3 domain binds to a C-terminal Arg SH3-binding site, and that an N-terminal ArgBP1 proline-rich sequence binds to the Arg SH3 domain. Immunostaining indicated that the subcellular localization of ArgBP1 is cytoplasmic. The similarity of the ArgBP1 expression pattern and subcellular localization to those of Arg and the potential for a highly specific and potentially strong association mediated by two pairs of SH3 domain/proline-rich motif interactions, suggest that ArgBP1 is likely to be a regulator and/or effector of Arg function.

• Kawamukai M
• [Adenylyl cyclase associated protein (CAP) and their homologs]
• Seikagaku. 1996; 68: 31-5

• Chen HI, Sudol M
• The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules.
• Proc Natl Acad Sci U S A. 1995; 92: 7819-23
• Display abstract

• The WW domain has previously been described as a motif of 38 semiconserved residues found in seemingly unrelated proteins, such as dystrophin, Yes-associated protein (YAP), and two transcriptional regulators, Rsp-5 and FE65. The molecular function of the WW domain has been unknown until this time. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP, which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous region consisting of a proline-rich domain followed by a tyrosine residue (with the shared sequence PPPPY), which we shall call the PY motif. Binding assays and site-specific mutagenesis have shown that the PY motif binds with relatively high affinity and specificity to the WW domain of YAP, with the preliminary consensus XPPXY being critical for binding. Herein, we have implicated the WW domain with a role in mediating protein-protein interactions, as a variant of the paradigm set by Src homology 3 domains and their proline-rich ligands.

• Kini RM, Evans HJ
• A hypothetical structural role for proline residues in the flanking segments of protein-protein interaction sites.
• Biochem Biophys Res Commun. 1995; 212: 1115-24
• Display abstract

• An examination of more than 1600 protein-protein interaction sites indicated that proline is the residue most commonly found near interaction sites. A structural role is distinguished for these proline residues in the flanking segments of protein-protein interaction sites. The unique nature of proline helps protect the integrity and present the sites, thus promoting protein-protein interactions. A novel approach to the design and development of potent peptide drugs and a simple predictive method to identify protein-protein interaction sites directly from the amino acid sequence have been developed based on this finding. The recognition of this structural role for proline has strong implications for protein chemistry and protein engineering.

• Sudol M
• Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product.
• Oncogene. 1994; 9: 2145-52
• Display abstract

• Yes belongs to the Src family of protein-tyrosine kinases. In order to understand molecular aspects of its signaling, we decided to isolate proteins that bind to the modulatory region of the Yes molecule. By generating anti-idiotypic antibodies against the aminoterminal domain of the Yes protein, we have identified, characterized and cloned a cDNA for a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. The protein is of 65 kiloDalton (kDa) molecular mass, it is phosphorylated in vivo on serine and is particularly rich in proline. We named it YAP65 for Yes-Associated Protein of 65 kDa. Within the YAP65 sequence, we identified a motif, PVKQPPPLAP, similar to that found in proteins that bind to the SH3 domain of the Abl kinase. Competition assays with synthetic peptides showed the involvement of the predicted proline-rich sequence in binding between YAP65 and the Yes kinase. The YAP65 protein was also shown to bind to other signaling molecules that contain SH3 domains including Nck, Crk and Src. At lower stoichiometry, YAP65 was also shown to bind to the SH3 domains of Abl and guanosine triphosphatase activating protein (GAP). Further characterization of YAP65 should illuminate Yes signaling pathways and could also identify a novel link between protein-tyrosine and serine kinases.

• Johnson DR, Bhatnagar RS, Knoll LJ, Gordon JI
• Genetic and biochemical studies of protein N-myristoylation.
• Annu Rev Biochem. 1994; 63: 869-914

• Morton CJ, Campbell ID
• SH3 domains. Molecular 'Velcro'.
• Curr Biol. 1994; 4: 615-7

• Mann DM, Romm E, Migliorini M
• Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.
• J Biol Chem. 1994; 269: 23661-7
• Display abstract

• Lactoferrin is an iron-binding protein which is synthesized by mucosal epithelium and neutrophils and released by these cells in response to inflammatory stimuli. It promotes neutrophil aggregation and manifests iron-dependent and -independent antimicrobial properties in vitro. Since lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polysaccharides can inhibit its clearance in vivo and in vitro, we sought to examine its interaction with the GAGs chondroitin sulfate and heparin. Amino-terminal sequencing of proteolytic fragments of human lactoferrin that were fractionated by GAG chromatography suggested that the amino-terminal 6 kDa of the secreted protein mediates its interaction with GAGs. Synthetic peptides were used to show that the first 33 residues of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin with an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed from this peptide, it then bound poorly to heparin (IC50 = 49 microM). Our results suggest that the GRRRRS sequence at the amino terminus of human lactoferrin acts synergistically with an RKVR sequence at positions 28-31 to form the predominate functional GAG-binding site of human lactoferrin. Molecular modeling of the crystalline structure of lactoferrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded protein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.

• Kim S, Willison KR, Horwich AL
• Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains.
• Trends Biochem Sci. 1994; 19: 543-8
• Display abstract

• CCT (also called the TCP-1 complex or TriC) is a chaperonin found in the eukaryotic cytosol, and has unique structural and functional features. Unlike homo-oligomeric chaperonins, CCT comprises at least eight different subunits, and appears to have a limited range of physiological substrates. We have analysed CCT sequences in light of the recent determination of the crystal structure and mutational identification of the functional domains of the bacterial chaperonin GroEL. A high level of identity among all chaperonin subunits is observed in those regions that correspond to the ATP-binding site of GroEL. By contrast, no significant identity is shared in the region corresponding to the polypeptide-binding region of GroEL, either between CCT subunits or between CCT subunits and GroEL. This suggests that the polypeptide-binding sites of CCT subunits have diverged both from each other and from GroEL, which may explain the apparently different range of substrates recognized by CCT.

• Peifer M, Berg S, Reynolds AB
• A repeating amino acid motif shared by proteins with diverse cellular roles.
• Cell. 1994; 76: 789-91

• Ordway RW, Pallanck L, Ganetzky B
• A TPR domain in the SNAP secretory proteins.
• Trends Biochem Sci. 1994; 19: 530-1

• Kopp M, Nwaka S, Holzer H
• Corrected sequence of the yeast neutral trehalase-encoding gene (NTH1): biological implications.
• Gene. 1994; 150: 403-4
• Display abstract

• We have identified a sequencing error in the neutral trehalase-encoding gene NTH1 [Kopp et al., J. Biol. Chem. 268 (1993) 4766-4774]. This error extends the deduced amino acid (aa) sequence at the N terminus by 58 aa. The biological implications of this include the presence of an additional phosphorylation site, which is believed to regulate trehalose hydrolysis.

• Birney E, Kumar S, Krainer AR
• A putative homology of U2AF65 in S. cerevisiae.
• Nucleic Acids Res. 1993; 21: 1333-1333

• Brown SC, Lucy JA
• Dystrophin as a mechanochemical transducer in skeletal muscle.
• Bioessays. 1993; 15: 413-9
• Display abstract

• This review is primarily concerned with two key issues in research on dystrophin: (1) how the protein interacts with the plasma membrane of skeletal muscle fibres and (2) how an absence of dystrophin gives rise to Duchenne muscular dystrophy. In relation to the first point, we suggest that the post-translational acylation of dystrophin may contribute to its interaction with the plasma membrane. Regarding the second point, it is generally considered that an absence of dystrophin makes the plasma membrane susceptible to damage by contraction/relaxation cycles. In this connection, we propose that the progressive nature of Duchenne dystrophy, and the phenotypic characteristics of mdx mice, are more consistent with dystrophin functioning as a mechanical transducer that transmits growth stimuli from the enlarging skeleton to the muscle. On the basis of this hypothesis, dystrophin-deficient muscles would be unable to grow at the same rate as the skeleton.

• Sapolsky RJ, Brendel V, Karlin S
• A comparative analysis of distinctive features of yeast protein sequences.
• Yeast. 1993; 9: 1287-98
• Display abstract

• The recently published sequence of yeast chromosome III (YCIII) provides the longest continuous stretch of a eukaryotic DNA molecule sequenced to date (315 kb). The sequence contains 116 distinct AUG-initiated open reading frames of at least 200 codons in length, more than 50 of which had not been described previously nor bear significant similarity to known proteins. We have analysed the YCIII known and putative protein sequences with respect to significant statistical features which might reflect on structural and functional characteristics. The YCIII proteins have striking similarities and differences in their sequence attribute distributions compared to the corresponding distributions for all available yeast sequences and other protein collections. Nine examples of YCIII proteins with distinctive sequence features are discussed in detail.

• Teunissen AW, Holub E, van der Hucht J, van den Berg JA, Steensma HY
• Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae.
• Yeast. 1993; 9: 423-7
• Display abstract

• The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A. and Steensma, H.Y. (1993). Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced. The sequence contains a large open reading frame of 2685 bp. The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus. Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.

• Veerkamp JH, van Kuppevelt TH, Maatman RG, Prinsen CF
• Structural and functional aspects of cytosolic fatty acid-binding proteins.
• Prostaglandins Leukot Essent Fatty Acids. 1993; 49: 887-906

• de Virgilio C, Burckert N, Neuhaus JM, Boller T, Wiemken A
• CNE1, a Saccharomyces cerevisiae homologue of the genes encoding mammalian calnexin and calreticulin.
• Yeast. 1993; 9: 185-8

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