Regulation of Bin1 SH3 domain binding by phosphoinositides

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.     

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.     

Regulation of PKC alpha activity by C1-C2 domain interactions

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.     

The identification of conserved interactions within the SH3 domain by alignment of sequences and structures

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein-protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved beta-bulge was identified in the second beta-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.     

The Ig-like domain of tapasin influences intermolecular interactions

Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.     

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.     

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance

Nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled-coil (CC) NLR that confers broad-spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell-death-inducing activity, whereas the CC-containing truncations, including CC-NB and CC-NB-LRR, do not induce cell-death responses. Coimmunoprecipitation (Co-IP) assay showed that the eCC domain self-associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V-mediated full-length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell-death induction. Two mutations (R73Q and E80K) interfered with D491V-mediated Pm21 autoactivation without affecting the cell-death-inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell-death-inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell-death signalling and disease-resistance signalling, which are potentially independent of each other.     

Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.     

Conserved patterns and interactions in the unfolding transition state across SH3 domain structural homologues

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long‐range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures.     

Overexpression of phospholipase C-gamma1 inhibits NGF-induced neuronal differentiation by proliferative activity of SH3 domain

Since the biological role of phospholipase C (PLC) gamma1 in neuronal differentiation still barely understood, here, we report that overexpression of PLC gamma1 inhibits neurite outgrowth and prolonged proliferation ability of PLC gamma1 contribute to the alteration of cell cycle regulatory proteins, subsequently exiting from cell growth arrest. Deletion of the SH3 or the entire SH223 domains, but not deletion of the N-SH2 or both the N-SH2 and C-SH2 domains expressing cells abolishes the differentiation-inhibitory effects of PLC gamma1, displaying depression of PCNA and elevation of cyclin D1. Moreover, these cells declined CDK1 and CDK2 expression and increased p21WAF-1, accompanying with G2/M accumulation. Some antiproliferative reagents are able to restore neurite outgrowth in PLC gamma1 cells, showing G2/M arrest. Our findings suggest that the proliferation activity of PLC gamma1 via its SH3 domain may be coupled with the flight from growth arrest by NGF, thereby inhibiting neuronal differentiation.     

BetaPix-a enhances the activity of phospholipase Cgamma1 by binding SH3 domain in breast cancer

Phospholipase C-gamma1 (PLCgamma1) plays a critical role in cell growth and proliferation by generating the second messengers, diacylglycerol and 1, 4, 5-inositol triphosphate. To investigate the roles of Src homology domain 2 and domain 3 of PLCgamma1 in PLCgamma1-mediated cell signaling, we characterized some proteins binding to these domains in the MCF7 and MDA-MB-231 breast cancer cell lines. Of the several proteins that bind to glutathione-S-transferase-SH2/SH2/SH3, we identified an 85 kDa protein that binds to the SH3 domain of PLCgamma1 as the guanine nucleotide exchange factor, p21-activated protein kinase-interacting exchange factor-a (betaPix-a). BetaPix-a co-immunoprecipitated with PLCgamma1 in breast cancer tissues extracts and in MCF7 and MDA-MB-231 cell extracts. In addition, PDGF-stimulated PLCgamma1 activity was elevated in betaPix-a-overexpressing NIH3T3 cells. Our results suggest that betaPix-a binds to the Src homology domain 3 of PLCgamma1 and promotes tumor growth in breast cancer by enhancing the activity PLCgamma1.     

Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain

The presence of residual structure in the unfolded state of the N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) has been investigated using far- and near-UV circular dichroism (CD), fluorescence, and NMR spectroscopy. The unfolded (U(exch)) state of the drkN SH3 domain is significantly populated and exists in equilibrium with the folded (F(exch)) state under non-denaturing conditions near physiological pH. Denaturation experiments have been performed on the drkN SH3 domain in order to monitor the change in ellipticity, fluorescence intensity, and chemical shift between the U(exch) state and chemically or thermally denatured states. Differences between the unfolded and chemically or thermally denatured states highlight specific areas of residual structure in the unfolded state that are cooperatively disrupted upon denaturation. Results provide evidence for cooperative interactions in the unfolded state involving residues of the central beta-sheet, particularly the beta4 strand. Denaturation as well as hydrogen-exchange experiments demonstrate a non-native burial of the Trp ring within this "cooperative" core of the unfolded state. These findings support the presence of non-native hydrophobic clusters, organised by Trp rings, within disordered states.     

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton's tyrosine kinase

The SH3 domain of Bruton's tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and (15)N NMR relaxation measurements are all consistent with a monomer-dimer equilibrium with a dissociation constant on the order of 60 microM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk.     

Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation.

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.     

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.