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Gabriel Waksman

Publications and source records attributed to Gabriel Waksman.

At least 37 records · Page 2Linked to original sources
PubMed2004

Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter.

Haemophilus influenzae is an important human pathogen that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hia autotransporter is an adhesive protein that promotes adherence to respiratory epithelial cells. Hia adhesive activity resides in two homologous binding domains, called HiaBD1 and HiaBD2. These domains interact with the same host cell receptor, but bind with different affinities. In this report, we describe the crystal structure of the high-affinity HiaBD1 binding domain, which has a novel trimeric architecture with three-fold symmetry and a mushroom shape. The subunit constituents of the trimer are extensively intertwined. The receptor-binding pocket is formed by an acidic patch that is present on all three faces of the trimer, providing potential for a multivalent interaction with the host cell surface, analogous to observations with the trimeric tumor necrosis factor superfamily of proteins. Hia is a novel example of a bacterial trimeric adhesin and may be the prototype member of a large family of bacterial virulence proteins with a similar architecture.

Adhesins, Bacterial↗
PubMed2004

SH2 domains: role, structure and implications for molecular medicine.

Src homology 2 (SH2) domains are protein modules (of approximately 100 amino acids) found in many proteins involved in tyrosine kinase signalling cascades. Their function is to bind tyrosine-phosphorylated sequences in specific protein targets. Binding of an SH2 domain to its cognate tyrosine-phosphorylated target links receptor activation to downstream signalling, both to the nucleus to regulate gene expression and throughout the cytoplasm of the cell. This review recapitulates the roles that SH2 domains play in normal and diseased states, describes the successes of SH2 domain research in deciphering their mechanism of action, and provides an overview of the use of SH2 domains as structural templates for the design of inhibitor drugs.

Humans↗
PubMed2004

Structural biology of bacterial pathogenesis.

Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis. The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor. A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis. Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.

Adhesins, Bacterial↗
PubMed2003

Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101.

Type IV secretion systems mediate intercellular transfer of macro-molecules via a mechanism ancestrally related to that of bacterial conjugation machineries. TraC of the IncN plasmid pKM101 belongs to the VirB5 family of proteins, an essential component of most type IV secretion systems. Here, we present the structure of TraC. VirB5/TraC is a single domain protein, which consists of a three helix bundle and a loose globular appendage. Structure-based site-directed mutagenesis followed by functional studies indicates that VirB5 proteins participate in protein-protein interactions important for pilus assembly and function.

Amino Acid Sequence↗
PubMed2003

The tandem Src homology 2 domain of the Syk kinase: a molecular device that adapts to interphosphotyrosine distances.

Conformational flexibility is important for protein function. However, information on the range of conformations accessible to macromolecules in the unbound state is often difficult to obtain. By using the model system of the tandem Src homology 2 domain (i.e., two adjacent Src homology 2 domains) of the Syk kinase, we report a method combining calorimetric and crystallographic measurements that reveals the preexistence of a conformational equilibrium in the unbound state, and that shows that this equilibrium is crucial for function.

Amino Acid Sequence↗
PubMed2003

Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor.

Recruitment of the Src kinase to the activated form of the platelet-derived growth factor (PDGF) receptor involves recognition of a unique sequence motif in the juxtamembrane region of the receptor by the Src homology 2 (SH2) domain of the enzyme. This motif contains two phosphotyrosine residues separated by one residue (sequence pYIpYV where pY indicates a phosphotyrosine). Here, we provide the thermodynamic and structural basis for the binding of this motif by the Src SH2 domain. We show that the second phosphorylation event increases the free energy window for specific interaction and that the physiological target is exquisitely designed for the task of recruiting specifically an SH2 domain which otherwise demonstrates very little intrinsic ability to discriminate sequences C-terminal to the first phosphorylation event. Surprisingly, we show that water plays a role in the recognition process.

Arginine↗
PubMed2003

VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N-terminal domains exhibit a collection of rigid-body conformations. Nucleotide binding 'locks' the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide-dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Biochemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.

Adenosine Triphosphatases↗
PubMed2002

The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity.

The Escherichia coli Rep protein is a 3' to 5' SF1 DNA helicase required for replication of bacteriophage phiX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximately 130 degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximately 1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.

Adenosine Triphosphatases↗
PubMed2002

Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation.

Periplasmic chaperones direct the assembly of adhesive, multi-subunit pilus fibers that play critical roles in bacterial pathogenesis. Pilus assembly occurs via a donor strand exchange mechanism in which the N-terminal extension of one subunit replaces the chaperone G(1) strand that transiently occupies a groove in the neighboring subunit. Here, we show that the chaperone primes the subunit for assembly by holding the groove in an open, activated conformation. During donor strand exchange, the subunit undergoes a topological transition that triggers the closure of the groove and seals the N-terminal extension in place. It is this topological transition, made possible only by the priming action of the chaperone that drives subunit assembly into the fiber.

Bacterial Proteins↗
PubMed2002

Crystal structure of the anticoagulant slow form of thrombin.

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.

Anticoagulants↗
PubMed2002

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface.

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.

Binding Sites↗
PubMed2002

Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding.

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.

Binding Sites↗
PubMed2002

Molecular recognition by SH2 domains.

In this chapter, we have described the biophysical investigations which have dissected the mechanisms of SH2 domain function. Due to nearly a decade and a half of investigation on SH2 domains, much about their binding mechanism has been characterized. SH2 domains have been found to have a positively charged binding cavity, largely conserved between different SH2 domains, which coordinates binding of the pTyr in the target. The ionic interactions between this pocket and the pTyr, in particular, between Arg beta B5 and the phosphate, provide the majority of the binding energy stabilizing SH2 domain-target interactions. The specificity in SH2 domain-target interactions emanates most often from the interactions between the residues C-terminal to the pTyr in the target and the specificity determining residues in the C-terminal half of the SH2 domain. However, the interactions in the specificity determining region of SH2 domains are weak, and hence single SH2 domains show only a modest level of specificity for tyrosine phosphorylated targets. Greater specificity in SH2 domain-containing protein-tyrosine phosphorylated target interactions can be achieved by placing SH2 domains in tandem (as is often found) or possibly through specific localization of SH2 domain-containing proteins within the cell. Although a relatively good understanding of how SH2 domains function in isolation has been obtained, the ways in which SH2 domain binding is coupled to allosteric transmission of signals in larger SH2 domain-containing proteins are still not clear. Hence, the future should bring further investigations of the mechanisms by which SH2 domain ligation alters the enzymatic activity and cellular localization of SH2 domain-containing proteins.

Binding Sites↗
PubMed2002

Hijacking the host cell proteasome.

Uropathogenic Escherichia coli subvert host cell signaling mechanisms to induce cellular responses that facilitate bacterial invasion and colonization. A recent publication in the November 15 issue of Cell shows that the bacterium may accomplish such a feat by hijacking the proteasome machinery.

Bacterial Toxins↗
PubMed2002

Phage P4 origin-binding domain structure reveals a mechanism for regulation of DNA-binding activity by homo- and heterodimerization of winged helix proteins.

The origin-binding domain of the gpalpha protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gpalpha activity by the protein Cnr. We have determined the crystal structure of P4-OBD at 2.95 A resolution. The structure of P4-OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4-OBD subunit is the DNA-binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4-OBD dimer is suggested. P4-OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gpalpha dimer. P4-OBD dimerization is mediated by helices alpha1 and alpha3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA-binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo- or heterodimerization scaffold.

Amino Acid Sequence↗
PubMed2002

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection.

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.

Adhesins, Bacterial↗