An intramolecular interaction between SH2-kinase linker and kinase domain is essential for the catalytic activity of protein-tyrosine kinase-6

Protein-tyrosine kinase-6 (PTK6, also known as Brk) is a non-receptor tyrosine kinase that contains SH3, SH2, and catalytic (Kinase) domains. We have identified an intramolecular interaction between the linker (Linker) region connecting the SH2 and Kinase domains and the Kinase domain. Residue Trp-184 within the Linker region is essential for the Linker-Kinase interaction but not for the Linker-SH3 interaction. A recombinant PTK6 Kinase domain connected to the Linker region had catalytic activity in terms of autophosphorylation, phosphorylation of a PTK6 substrate, BKS, and phosphorylation of an oligopeptide substrate, whereas the Kinase domain itself, or one connected to a Linker region containing a W184A substitution, did not. The introduction of the W184A mutation into PTK6 also abrogated autophosphorylation and phosphorylation of another PTK6 substrate, Sam68, as well as phosphorylation of intracellular proteins. It also abolished the ability of PTK6 to promote proliferation and prevent apoptosis of HEK 293 cells, as well as to permit anchorage-independent colony formation. Therefore, unlike Src family members, in which the Linker-Kinase interaction inhibits catalytic activity, in PTK6 this interaction has an essential positive role.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.     

The SH3 domain of a M7 interacts with its C-terminal proline-rich region

Myosins play essential roles in migration, cytokinesis, endocytosis, and adhesion. They are composed of a large N-terminal motor domain with ATPase and actin binding sites and C-terminal neck and tail regions, whose functional roles and structural context in the protein are less well characterized. The tail regions of myosins I, IV, VII, XII, and XV each contain a putative SH3 domain that may be involved in protein-protein interactions. SH3 domains are reported to bind proline-rich motifs, especially "PxxP" sequences, and such interactions serve regulatory functions. The activity of Src, PI3, and Itk kinases, for example, is regulated by intramolecular interactions between their SH3 domain and internal proline-rich sequences. Here, we use NMR spectroscopy to reveal the structure of a protein construct from Dictyostelium myosin VII (DdM7) spanning A1620-T1706, which contains its SH3 domain and adjacent proline-rich region. The SH3 domain forms the signature beta-barrel architecture found in other SH3 domains, with conserved tryptophan and tyrosine residues forming a hydrophobic pocket known to bind "PxxP" motifs. In addition, acidic residues in the RT or n-Src loops are available to interact with the basic anchoring residues that are typically found in ligands or proteins that bind SH3 domains. The DdM7 SH3 differs in the hydrophobicity of the second pocket formed by the 3(10) helix and following beta-strand, which contains polar rather than hydrophobic side chains. Most unusual, however, is that this domain binds its adjacent proline-rich region at a surface remote from the region previously identified to bind "PxxP" motifs. The interaction may affect the orientation of the tail without sacrificing the availability of the canonical "PxxP"-binding surface.     

Dynamic interaction of CD2 with the GYF and the SH3 domain of compartmentalized effector molecules

Intracellular protein interaction domains are essential for eukaryotic signaling. In T cells, the CD2BP2 adaptor binds two membrane-proximal proline-rich motifs in the CD2 cytoplasmic tail via its GYF domain, thereby regulating interleukin-2 production. Here we present the structure of the GYF domain in complex with a CD2 tail peptide. Unlike SH3 domains, which use two surface pockets to accommodate proline residues of ligands, the GYF domain employs phylogenetically conserved hydrophobic residues to create a single interaction surface. NMR analysis shows that the Fyn but not the Lck tyrosine kinase SH3 domain competes with CD2BP2 GYF-domain binding to the same CD2 proline-rich sequence in vitro. To test the in vivo significance of this competition, we used co-immunoprecipitation experiments and found that CD2BP2 is the ligand of the membrane-proximal proline-rich tandem repeat of CD2 in detergent-soluble membrane compartments, but is replaced by Fyn SH3 after CD2 is translocated into lipid rafts upon CD2 ectodomain clustering. This unveils the mechanism of a switch of CD2 function due to an extracellular mitogenic signal.     

Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin.

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.     

The adaptor protein Gads (Grb2-related adaptor downstream of Shc) is implicated in coupling hemopoietic progenitor kinase-1 to the activated TCR

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.     

Solution structure of the tandem Src homology 3 domains of p47phox in an autoinhibited form

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O(2).) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).     

A novel, specific interaction involving the Csk SH3 domain and its natural ligand.

C-terminal Src kinase (Csk) takes part in a highly specific, high affinity interaction via its Src homology 3 (SH3) domain with the proline-enriched tyrosine phosphatase PEP in hematopoietic cells. The solution structure of the Csk-SH3 domain in complex with a 25-residue peptide from the Pro/Glu/Ser/Thr-rich (PEST) domain of PEP reveals the basis for this specific peptide recognition motif involving an SH3 domain. Three residues, Ala 40, Thr 42 and Lys 43, in the SH3 domain of Csk specifically recognize two hydrophobic residues, Ile 625 and Val 626, in the proline-rich sequence of the PEST domain of PEP. These two residues are C-terminal to the conventional proline-rich SH3 domain recognition sequence of PEP. This interaction is required in addition to the classic polyproline helix (PPII) recognition by the Csk-SH3 domain for the association between Csk and PEP in vivo. NMR relaxation analysis suggests that Csk-SH3 has different dynamic properties in the various subsites important for peptide recognition.     

Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

Structure, dynamics, and Hck interaction of full-length HIV-1 Nef

Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

A specific intermolecular association between the regulatory domains of a Tec family kinase

Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. We have examined a novel intermolecular interaction between the SH3 and SH2 domains of Itk. In addition to the interaction between the isolated domains, we have found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that we describe provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, we note a striking correlation between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agamaglobulinemia.     

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.     

Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.     

Solution structure of Grb2 reveals extensive flexibility necessary for target recognition

Grb2 is an adaptor protein composed of a single SH2 domain flanked by two SH3 domains. Grb2 functions as an important evolutionary conserved link between a variety of cell membrane receptors and the Ras/MAP kinase-signaling cascade. Here, we describe the solution structure of Grb2 as revealed by NMR and small angle X-ray scattering measurements. We demonstrate that Grb2 is a flexible protein in which the C-terminal SH3 domain is connected to the SH2 domain via a flexible linker. This is in contrast to the previously described Grb2 crystal structure, which showed a compact structure with intramolecular contact between two SH3 domains. Binding experiments on Grb2 and peptides containing two different proline-rich sequences indicate that Grb2 adapts the relative position and orientation of the two SH3 domains to bind bivalently to the target peptide sequences.     

Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis.

The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255-pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.     

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.