Search PubMed⌕ Search

Biomedical subjects

D Cowburn

Publications and source records attributed to D Cowburn.

At least 37 records · Page 2Linked to original sources
PubMed1998

Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus.

A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.

Amino Acid Sequence↗
PubMed1998

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The solution structure and dynamics of the pleckstrin homology domain of G protein-coupled receptor kinase 2 (beta-adrenergic receptor kinase 1). A binding partner of Gbetagamma subunits.

The solution structure of an extended pleckstrin homology (PH) domain from the beta-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the beta-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal alpha-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended alpha-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gbetagamma subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains.

Amino Acid Sequence↗
PubMed1997

The solution structure of the pleckstrin homology domain of human SOS1. A possible structural role for the sequential association of diffuse B cell lymphoma and pleckstrin homology domains.

A large subset of pleckstrin homology (PH) domains are immediately to the C terminus of diffuse B cell lymphoma (Dbl) homology (DbH) domains. Dbl domains are generally considered to be GTPase-exchange factors; many are proto-oncogenes. PH domains appear to function as membrane-recruitment factors, or have specific protein-protein interactions. Since dual domain (DbH/PH) constructs are known to have significant properties in other pathways, it is possible that a defined interdomain relationship is required for DbH/PH function. We determined the solution structure of the human SOS1 PH domain for a construct partially extended into the preceding DbH domain. There are specific structural contacts between the PH and the vestigial DbH domain. This appears to involve structural elements common to this subfamily of PH domains, and to DbH domains. The human SOS1 PH domain binds to inositol 1,4,5-triphosphate with a approximately 60 mu M affinity. Using chemical shift titration, the binding site is identified to be essentially identical to that observed crystallographically for the inositol 1,4,5-triphosphate complex with the PH domain of phospholipase Cdelta. This site may serve as an interdomain regulator of DbH or other domains' functions. While the overall fold of the human SOS1 PH domain is similar to other PH domains, the size and position of the intrastrand loops and the presence of an N-terminal alpha-helix of the vestigial DbH domain suggest that the subfamily of PH domains associated with DbH domains may be a well defined structural group in which the PH domain is a membrane recruiter and modulator.

Amino Acid Sequence↗
PubMed1997

The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration.

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.

Blood Proteins↗
PubMed1997

Peptide recognition by PTB and PDZ domains.

Protein tyrosine binding (PTB) and 'post synaptic density disc-large zo-1' (PDZ) domains bind to short peptidic ligands by augmentation of one of the domain's beta sheets and other recognition mechanisms. The two domain classes have a superficial resemblance to each other, even though no sequential homology exists. The structural bases of the interactions are well understood for the few domains now experimentally determined, and ligand-target pairs can probably be identified in favorable cases by analogy with the known domains. For both PTB and PDZ classes, functional activities are still not fully defined: it is possible that these domain classes, along with pleckstrin homology domains, have multiple roles.

Amino Acid Sequence↗
PubMed1997

Modular peptide recognition domains in eukaryotic signaling.

A characteristic feature of cellular signal transduction pathways in eukaryotes is the separation of catalysis from target recognition. Several modular domains that recognize short peptide sequences and target signaling proteins to these sequences have been identified. The structural bases of the specificities of recognition by SH2, SH3, and PTB domains have been elucidated by X-ray crystallography and NMR, and these results are reviewed here. In addition, the mechanism of cooperative interactions between these domains is discussed.

Amino Acid Sequence↗
PubMed1996

Adaptors and integrators.

The PTB domain expands both the PH-domain set and peptide-protein recognition motifs; the PDZ domain shows an intriguing resemblance.

Binding Sites↗
PubMed1996

Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.

It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.

Binding Sites↗
PubMed1996

Much more than the sum of the parts: structures of the dual SH2 domains of ZAP-70 and Syp.

Proteins involved in signaling pathways frequently contain one or more SH2 domains. New structural information on proteins that carry two SH2 domains show, surprisingly, that the domains are closely interlinked, so the binding sites are rigidly oriented with respect to each other. Thus, only ligands with the right spacing of the phosphotyrosines will be tightly bound.

Intracellular Signaling Peptides and Proteins↗
PubMed1995

Enhanced affinities and specificities of consolidated ligands for the Src homology (SH) 3 and SH2 domains of Abelson protein-tyrosine kinase.

The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins.

Abelson murine leukemia virus↗
PubMed1995

A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.

Amino Acid Sequence↗
PubMed1995

The solution structure of Abl SH3, and its relationship to SH2 in the SH(32) construct.

BACKGROUND: The Src homology domains, SH3 and SH2, of Abl protein tyrosine kinase regulate enzymatic activity in vivo. Abl SH3 suppresses kinase activity, whereas Abl SH2 is required for the transforming activity of the activated form of Abl. We expect that the solution structures of Abl SH3, Abl SH2 and Abl SH(32) (a dual domain comprising SH3 and SH2 subdomains) will contribute to a structural basis for understanding the mechanism of the Abl 'regulatory apparatus'. RESULTS: We present the solution structure of the free Abl SH3 domain and a structural characterization of the Abl regulatory apparatus, the SH(32) dual domain. The solution structure of Abl SH3 was determined using multidimensional double resonance NMR spectroscopy. It consists of two antiparallel beta sheets packed orthogonally, an arrangement first shown in spectrin SH3. Compared with the crystal structure of the Abl SH3 complexed with a natural ligand, there is no significant difference in overall folding pattern. The structure of the Abl SH(32) dual domain was characterized by NMR spectroscopy using the 1H and 15N resonance assignment of Abl SH3 and Abl SH2. On the basis of the high degree of similarity in chemical shifts and hydrogen/deuterium exchange pattern for the individual domains of SH3 and SH2 compared with those of the SH(32) dual domain, a structural model of the Abl SH(32) regulatory apparatus is suggested. This model is in good agreement with the ligand-binding characteristics of Abl SH3, SH2 and SH(32). The binding constants for isolated SH3 and SH2 domains when binding to natural ligands, measured by intrinsic fluorescence quenching, do not differ significantly from the constants of these domains within SH(32). CONCLUSION: The solution structures of free Abl SH3 and Abl SH2, and the structural model of Abl SH(32), provide information about the overall topology of these modular domains. The structural model of Abl SH(32), a monomer, consists of the SH3 and SH2 domains connected by a flexible linker. Sites of ligand binding for the two subdomains are independent.

Amino Acid Sequence↗
PubMed1995

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

BACKGROUND: Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS: In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Amino Acid Sequence↗