Structural basis of PxxDY motif recognition in SH3 binding

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

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.     

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.     

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

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

Propensity for C-terminal domain swapping correlates with increased regional flexibility in the C-terminus of RNase A

Domain swapping is a type of oligomerization in which monomeric proteins exchange a structural element, resulting in oligomers whose subunits recapitulate the native, monomeric fold. It has been implicated as a potential mechanism for protein aggregation, which provides a strong impetus to understand the structural determinants and folding mechanisms that trigger domain swapping. Bovine pancreatic ribonuclease A (RNase A) is a well-studied protein known to domain swap under extreme conditions, such as lyophilization from acetic acid. The major domain-swapped dimer form of RNase A exchanges a β-strand at its C-terminus to form a C-terminal domain-swapped dimer. To study the mechanism by which C-terminal swapping occurs, we used a variant of RNase A containing a P114G mutation that readily domain swaps under physiological conditions. Using NMR and hydrogen-deuterium exchange, we find that the P114G variant has decreased protection from hydrogen exchange compared to the wild-type protein near the C-terminal hinge region. Our results suggest that domain swapping occurs via a local high-energy fluctuation at the C-terminus.     

A novel peptide-SH3 interaction.

SH3 domains constitute a family of protein-protein interaction modules that bind to peptides displaying an X-proline-X-X-proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non-receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage-displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross-linking, we demonstrate that a proline-X-X-aspartate-tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8-related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3-containing proteins that do not bind to canonical XPXXP-containing peptides, and that establish distinct interactions in the signaling network.     

Determinants of the SRC homology domain 3-like fold

In eukaryotes, the Src homology domain 3 (SH3) is a very important motif in signal transduction. SH3 domains recognize poly-proline-rich peptides and are involved in protein-protein interactions. Until now, the existence of SH3 domains has not been demonstrated in prokaryotes. However, the structure of the C-terminal domain of DtxR clearly shows that the fold of this domain is very similar to that of the SH3 domain. In addition, there is evidence that the C-terminal domain of DtxR binds to poly-proline-rich regions. Other bacterial proteins have domains that are structurally similar to the SH3 domain but whose functions are unknown or differ from that of the SH3 domain. The observed similarities between the structures of the C-terminal domain of DtxR and the SH3 domain constitute a perfect system to gain insight into their function and information about their evolution. Our results show that the C-terminal domain of DtxR shares a number of conserved key hydrophobic positions not recognizable from sequence comparison that might be responsible for the integrity of the SH3-like fold. Structural alignment of an ensemble of such domains from unrelated proteins shows a common structural core that seems to be conserved despite the lack of sequence similarity. This core constitutes the minimal requirements of protein architecture for the SH3-like fold.     

Structural studies of the Cpx pathway activator NlpE on the outer membrane of Escherichia coli

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In Escherichia coli, adhesion to abiotic surfaces has been reported to activate the Cpx pathway in an NlpE-dependent manner. External copper ions are also thought to activate the Cpx pathway mediated by NlpE. We determined the crystal structure of NlpE from E. coli at 2.6 A resolution. The structure showed that NlpE consists of two beta barrel domains. The N-terminal domain resembles the bacterial lipocalin Blc, and the C-terminal domain has an oligonucleotide/oligosaccharide-binding (OB) fold. From both biochemical analyses and the crystal structure, it can be deduced that the cysteine residues in the CXXC motif may be chemically active. Furthermore, two monomers in the asymmetric unit form an unusual 3D domain-swapped dimer. These findings indicate that tertiary and/or quaternary structural instability may be responsible for Cpx pathway activation.     

Three-dimensional domain swapping in the protein structure space

Since the proposal of three-dimensional (3D) domain swapping, many 3D domain-swapped structures have been reported. However, when compared with the vast protein structure space, it is still unclear whether 3D domain swapping is a general mechanism for protein assembly. Here, we investigated this possibility by constructing a dataset consisting of more than 500 domain-swapped structures. The domain-swapped structures were mapped into the protein structure space. We found that about 10% of protein folds and 5% of protein families contain domain-swapped structures. When comparing the domain-swapped structures in a family/superfamily, we found that proteins within a family/superfamily can swap in different ways. Interface analysis revealed that the hinge loops contributed more than half of the open interface in 70% of bona fide domain-swapped dimers, indicating that the hinge loops play an important role in stabilizing the domain-swapped conformations. Our study supports the suggestion that domain swapping is a general property of all proteins and will facilitate further understanding the mechanism of 3D domain swapping.     

Solution structure of Eps15's third EH domain reveals coincident Phe-Trp and Asn-Pro-Phe binding sites

Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.     

Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding

ALIX/AIP1 functions in enveloped virus budding, endosomal protein sorting, and many other cellular processes. Retroviruses, including HIV-1, SIV, and EIAV, bind and recruit ALIX through YPX(n)L late-domain motifs (X = any residue; n = 1-3). Crystal structures reveal that human ALIX is composed of an N-terminal Bro1 domain and a central domain that is composed of two extended three-helix bundles that form elongated arms that fold back into a "V." The structures also reveal conformational flexibility in the arms that suggests that the V domain may act as a flexible hinge in response to ligand binding. YPX(n)L late domains bind in a conserved hydrophobic pocket on the second arm near the apex of the V, whereas CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain, and both interactions are required for virus budding. ALIX therefore serves as a flexible, extended scaffold that connects retroviral Gag proteins to ESCRT-III and other cellular-budding machinery.     

Intertwined dimeric structure for the SH3 domain of the c-Src tyrosine kinase induced by polyethylene glycol binding

Here we report the first crystal structure of the SH3 domain of the cellular Src tyrosine kinase (c-Src-SH3) domain on its own. In the crystal two molecules of c-Src-SH3 exchange their -RT loops generating an intertwined dimer, in which the two SH3 units, preserving the binding site configuration, are oriented to allow simultaneous binding of two ligand molecules. The dimerization of c-Src-SH3 is induced, both in the crystal and in solution, by the binding of a PEG molecule at the dimer interface, indicating that this type of conformations are energetically close to the native structure. These results have important implications respect to in vivo oligomerization and amyloid aggregation.     

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.     

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.     

The switch helix: A putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase

For over three decades the isozymes of cGMP-dependent protein kinase (PKG) have been studied using an array of biochemical and biophysical techniques. When compared to its closest cousin, cAMP-dependent protein kinase (PKA), these studies revealed a set of identical domain types, yet containing distinct, sequence-specific features. The recently solved structure of the PKG regulatory domain showed the presence of the switch helix (SW), a novel motif that promotes the formation of a domain-swapped dimer in the asymmetric unit. This dimer is mediated by the interaction of a knob motif on the C-terminal locus of the SW, with a hydrophobic nest on the opposing protomer. This nest sits adjacent to the cGMP binding pocket of the B-site. Priming of this site by cGMP may influence the geometry of the hydrophobic nest. Moreover, this unique interaction may have wide implications for the architecture of the inactive and active forms of PKG. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Structure of the monomeric 8-kDa dynein light chain and mechanism of the domain-swapped dimer assembly

The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal beta-strand (the beta2-strand) at neutral pH. The protein undergoes non-reversible dimer-to-monomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the beta2-strand of the protein, which undergoes transition from a beta-strand to a nascent alpha-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight beta1/beta2-loop dramatically stabilized the monomer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The three-dimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the beta2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping.     

An extended hydrophobic core induces EF-hand swapping

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.     

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.     

An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.

KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5'-TTTAGC(G/C)GCTAAA-3') present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297-358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains.     

Eps8 in the midst of GTPases

Eps8, originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR), displays a domain organization typical of a signaling molecule that includes a putative N-terminal PTB domain, a central SH3 domain, and a C-terminal "effector region". This latter region directs Eps8 localization within the cell and is sufficient to activate the GTPase, Rac, leading to actin cytoskeletal remodeling. Eps8 binds, through its SH3 domain, to either Abi1 (also called E3b1) or RN-tre. Abi1 scaffolds together Eps8 and Sos1, a dual specificity guanine nucleotide exchange factor for Ras and Rac proteins, thus facilitating the formation of a trimeric complex, in turn required for activation of Rac. On the other hand, RN-tre, a Rab5 GTPase activating protein, by entering in a complex with Eps8, inhibits EGFR internalization. Furthermore, RN-tre competes with Abi1 for binding to Eps8, diverting the latter from its Rac-activating function. Thus, depending on its engagement in different complexes, Eps8 participates to EGFR signaling through Rac and endocytosis through Rab5.