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.     

Solution structure of the PX domain, a target of the SH3 domain

The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.     

Solution structure of the SH3 domain of DOCK180

DOCK180 family proteins are Rho guanine nucleotide exchange factors. DOCK1‐5 contains an N‐terminal SH3 domain implicated in their autoinhibition. Release of the closed conformation requires the interaction between SH3 and engulfment and cell motility (ELMO). Here, we solved the solution structure of DOCK180 SH3 domain, which shares similar target binding features with the SH3 domain of DOCK2. The conserved N‐terminal extension packs with the SH3 core domain and forms a new target binding site distinct from the canonical “PxxP” site. Our results demonstrate that the bidentate target binding mode of DOCK180 SH3 domain might be a general feature in all DOCK proteins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis

Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N-Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N-Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent-exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context-dependent effect resulting from an interaction between neighboring charged side-chains.     

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized. Several studies suggest that the C-terminal SH3 domain plays an important regulatory role in the protein; however, no structural information is available on this domain, and relatively little is known about its binding partners. In the current work, we have solved the solution NMR structure of the C-terminal SH3 domain. The domain adopts the standard SH3 fold comprising a five-stranded beta barrel. In agreement with alignment and modeling studies, the structure indicates that the canonical-binding surface of the SH3 domain is unusually polar and suggests that this domain may not bind typical PXXP ligands or that it may bind them with reduced affinity. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. These studies offer some insight into the likely structural and thermodynamic consequences of point mutations in the cSH3 domain that are known to deregulate Crk-II function. Our results set the stage for a better understanding the role of the cSH3 domain in the context of the full-length protein.     

Solution structure of the first SH3 domain of human vinexin and its interaction with vinculin peptides

Solution structure of the first Src homology (SH) 3 domain of human vinexin (V_SH3_1) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical beta-beta-beta-beta-alpha-beta fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V_SH3_1. The interaction between P868 and V_SH3_1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V_SH3_1 had a low binding affinity. The structure and ligand-binding interface of V_SH3_1 provide a structural basis for the further functional study of this important molecule.     

Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phosphorylation of a common site in CD3epsilon

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3epsilon to Eps8L1 and Nck SH3 domains. CD3epsilon bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3epsilon to interact with either SH3 or SH2 domain-containing proteins.     

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.     

Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes

We studied the interaction of hematopoietic cell kinase SH3 domain (HckSH3) with an artificial 12-residue proline-rich peptide PD1 (HSKYPLPPLPSL) identified as high affinity ligand (K(D)=0.2 muM). PD1 shows an unusual ligand sequence for SH3 binding in type I orientation because it lacks the typical basic anchor residue at position P(-3), but instead has a tyrosine residue at this position. A basic lysine residue, however, is present at position P(-4). The solution structure of the HckSH3:PD1 complex, which is the first HckSH3 complex structure available, clearly reveals that the P(-3) tyrosine residue of PD1 does not take the position of the typical anchor residue but rather forms additional van der Waals interactions with the HckSH3 RT loop. Instead, lysine at position P(-4) of PD1 substitutes the function of the P(-3) anchor residue. This finding expands the well known ligand consensus sequence +xxPpxP by +xxxPpxP. Thus, software tools like iSPOT fail to identify PD1 as a high-affinity HckSH3 ligand so far. In addition, a short antiparallel beta-sheet in the RT loop of HckSH3 is observed upon PD1 binding. The structure of the HckSH3:PD1 complex reveals novel features of SH3 ligand binding and yields new insights into the structural basics of SH3-ligand interactions. Consequences for computational prediction tools adressing SH3-ligand interactions as well as the biological relevance of our findings are discussed.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Solution structure of a SRP19 binding domain in human SRP RNA

Assembly of the human signal recognition particle (SRP) requires SRP19 protein to bind to helices 6 and 8 of SRP RNA. In the present study, structure of a 29-mer RNA composing the SRP19 binding site in helix 6 was determined by NMR spectroscopy. The two A:C mismatches were continuously stacked to each other and formed wobble type A:C base pairs. The GGAG tetraloop in helix 6 was found to adopt a similar conformation to that of GNRA tetraloop, suggesting that these tetraloops are included in an extensive new motif GNRR. Compared with the crystal structure of helix 6 in complex with SRP19 determined previously, the GGAG tetraloop in the complex was found to adopt a similar conformation to the free form, although the loop structure becomes more open upon SRP19 binding. Thus, SRP19 is thought to recognize the overall fold of the GGAG loop.     

Diversity in the SH2 domain family phosphotyrosyl peptide binding site

Src homology 2 (SH2) domains are approximately 100 residue phosphotyrosyl peptide binding modules found in signalling proteins and are important targets for therapeutic intervention. The peptide binding site is evolutionarily well conserved, particularly at the two major binding pockets, pTyr and pTyr + 3. We present a computational analysis of diversity within the peptide binding region and discuss molecular recognition beyond the conventional binding motif, drawing attention to novel conserved ligand interaction sites which may be exploitable in ligand binding studies. The peptide binding site is defined by selecting crystal contacts and domains are clustered according to binding site residue similarity. Comparison with a classification based on experimental peptide screening reveals a high level of qualitative agreement, indicating that the method is able independently to generate functional information. A conservation scoring method reveals extensive patches of conservation in some groups not present across the whole family, challenging the notion that the domains recognise only a linear phosphopeptide sequence. Conservation difference maps determine group-dependent clusters of conserved residues that are not seen when considering a larger experimentally determined group. Many of these residues contact the peptide outside the pTyr to pTyr + 3 motif, challenging the conventional view that this motif is largely responsible for ligand recognition and discrimination.     

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).     

Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme

A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.     

Nephrectomy and a newly identified binding site for angiotensin II in the rat adrenal cortex.

Angiotensin II (Ang II) binding sites in adrenal glands of nephrectomized rats were investigated by in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as ligands. Ang II binding site was increased to 161% in the cortex and decreased to 67% in the medulla 48 h after nephrectomy. In the medulla, the AT2 antagonist (PD123177, 5 microM) inhibited specific binding by 90% whereas the AT1 antagonist (DuP753, 5 microM) inhibited by only 10%. In contrast, in the cortex, neither DuP753 (5 microM) nor PD123177 (5 microM) substantially inhibited the binding. Binding in the presence of either the AT1 or AT2 antagonist was abolished by the simultaneous presence of both antagonists. These results suggest the presence of a new Ang II binding site with unique pharmacological properties and differing from currently known subtypes of Ang II receptors, in the adrenal cortex after nephrectomy.     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.     

Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lck SH3 ligands using phage display. In addition, we analyzed Lck SH3 binding sites within known natural Lck SH3 binding proteins using an Lck specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lck SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lck SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands-which are not due to significant K(D) value differences as shown by calculating Lck SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lck SH3 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions.     

Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein.

The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.