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A M Stock

Publications and source records attributed to A M Stock.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis and biochemical characterization of a phosphorylated analogue of the response regulator CheB.

CheB is a response regulator protein in the bacterial chemotaxis two-component signal transduction pathway. Methylesterase CheB functions together with methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli. The level of glutamate methylation in turn indirectly controls the direction of flagellar rotation. Like most two-component response regulators, CheB is activated in vivo by phosphorylation of a single aspartate, Asp 56, in its regulatory domain. Extensive biochemical and crystallographic studies have been completed on the inactive, unphosphorylated form of CheB. Because of the inherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isolated for further characterization. We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and chemical modification strategies. Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB. The unique cysteine in the substituted form of CheB was modified by sodium thiophosphate, Na(3)SPO(3), using two sequential disulfide bond exchange reactions. The analogue, D56C/C207S/C309S CheB-SPO(3), contained a thiophosphate group covalently bonded to the protein through a disulfide linkage at residue 56. Mass spectrometry showed that the protein was singly modified. Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that the analogue had a half-life of 28 days. In in vitro methylesterase assays in the presence of Mg(2+), the analogue exhibited activity equivalent to that of fully phosphorylated C207S/C309S CheB. Thus, D56C/C207S/C309S CheB-SPO(3) is a stable analogue that may be useful for characterization of the active form of CheB.

Aspartic Acid↗
PubMed2001

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB.

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.

Amides↗
PubMed2001

Histidine kinases and response regulator proteins in two-component signaling systems.

Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating histidine protein kinases provide phosphoryl groups for response regulator proteins which, in turn, function as molecular switches that control diverse effector activities. Structural studies of proteins involved in two-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.

Histidine Kinase↗
PubMed2000

Synthesis of [(32)P]phosphoramidate for use as a low molecular weight phosphodonor reagent.

Phosphoramidate serves as a useful phosphodonor reagent in protein and peptide phosphorylation, notably in studying two-component signal transduction systems in which low molecular weight phosphodonors can substitute for the phosphodonor function of histidine protein kinases in in vitro phosphorylation studies of response regulator proteins. A convenient method for the synthesis of radiolabeled phosphoramidate has not been developed, and this has limited its broader use. Here we report the synthesis of radiolabeled ammonium hydrogen phosphoramidate [(NH(4))H(32)PO(3)NH(2)] which is achieved by activation of [(32)P]orthophosphate with ethyl isocyanate followed by aminolysis with ammonium hydroxide to form the desired phosphoramidate. The procedure is conveniently carried out in a microfuge tube and requires only two successive precipitation steps to obtain pure ammonium hydrogen phosphoramidate. Molar yields of 15-30% and specific activities of 10-20 Ci/mol are readily achieved. Phosphorylation of microgram quantities of response regulator proteins CheY, CheB, and DrrA is shown. Low level, but detectable, nonspecific phosphorylation was observed for reactions near ambient temperatures when substrate response regulators lacking the active site aspartate but containing histidine residues are used. More significant levels of nonspecific phosphorylation were observed for reactions at elevated temperatures when using a nonresponse regulator control protein (RNase A).

Amides↗
PubMed2000

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Camphor↗
PubMed2000

A tale of two components: a novel kinase and a regulatory switch.

Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways. Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes. Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains. Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue. The altered molecular surface promotes specific protein-protein interactions that mediate the downstream response.

Bacterial Proteins↗
PubMed2000

Two-component signal transduction.

Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.

Anti-Bacterial Agents↗
PubMed1999

The role of water in the catalytic efficiency of triosephosphate isomerase.

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.

Animals↗
PubMed1999

High energy exchange: proteins that make or break phosphoramidate bonds.

Several proteins that catalyze phosphoryl transfer reactions involving phosphohistidine residues have recently been structurally characterized. The architecture of two histidine kinases defines a new protein kinase fold. The diverse folds of several phosphotransfer proteins appear to be designed to foster protein-protein interactions between transfer partners.

Adenosine Triphosphate↗
PubMed1999

Orientation of OmpR monomers within an OmpR:DNA complex determined by DNA affinity cleaving.

Escherichia coli OmpR is a transcription factor that regulates the differential expression of the porin genes ompF and ompC. Phosphorylated OmpR binds as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 10-bp half-sites. Expression of the ompF gene is achieved by the hierarchical occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4. Despite genetic, biochemical, and structural studies, specific details of the interaction between phosphorylated OmpR and the DNA remain unknown. We have linked the DNA cleaving moiety o-phenanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in the OmpR:F1 complex. Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to construct a model of the OmpR:F1 complex. We propose that OmpR binds asymmetrically to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove. The N-terminal end of the recognition helix is promoter-proximal and flanked by "wings" W1 and W2 positioned proximally and distally, respectively, to the transcription start site of ompF. We further propose that the C-terminal end of the recognition helix makes the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix.

Bacterial Proteins↗
PubMed1998

Structural analysis of bacterial chemotaxis proteins: components of a dynamic signaling system.

Most motile bacteria are capable of directing their movement in response to chemical gradients, a behavior known as chemotaxis. The signal transduction system that mediates chemotaxis in enteric bacteria consists of a set of six cytoplasmic proteins that couple stimuli sensed by a family of transmembrane receptors to behavioral responses generated by the flagellar motors. Signal transduction occurs via a phosphotransfer pathway involving a histidine protein kinase, CheA, and a response regulator protein, CheY, that in its phosphorylated state, modulates the direction of flagellar rotation. Two auxiliary proteins, CheW and CheZ, and two receptor modification enzymes, methylesterase CheB and methyltransferase CheR, influence the flux of phosphoryl groups within this central pathway. This paper focuses on structural characteristics of the four signaling proteins (CheA, CheY, CheB, and CheR) for which NMR or x-ray crystal structures have been determined. The proteins are examined with respect to their signaling activities that involve reversible protein modifications and transient assembly of macromolecular complexes. A variety of data suggest conformational flexibility of these proteins, a feature consistent with their multiple roles in a dynamic signaling pathway.

Bacterial Physiological Phenomena↗
PubMed1998

Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima.

The central signaling pathway in many bacterial regulatory systems involves phosphotransfer between two conserved proteins, a histidine protein kinase, and a response regulator. The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how both the proteins and the labile acyl phosphate of the response regulator are adapted to function at elevated temperatures. Thermotoga maritima HpkA is a transmembrane histidine kinase, and DrrA is its cognate response regulator. Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized. HpkA57 and DrrA have apparent Tm's of 75 and 90 degreesC, respectively. HpkA57 exhibits ATP-dependent autophosphorylation activity similar to that of histidine kinases from mesophiles, with maximum activity at 70 degreesC. DrrA catalyzes transfer of phosphoryl groups from HpkA57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degreesC. At this temperature, the half-life for phospho-DrrA is approximately 3 min. In the absence of Mg2+, the half-life is 26 min, significantly greater than the half-life of a typical acyl phosphate at 80 degreesC. In the absence of Mg2+, at all temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate. This suggests that the active site of this hyperthermophilic response regulator is designed to protect the phospho-aspartyl residue from hydrolysis.

Aspartic Acid↗
PubMed1998

Activation of methylesterase CheB: evidence of a dual role for the regulatory domain.

The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level of chemoreceptor methylation, influencing the signaling activities of the receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-domain architecture consisting of an N-terminal regulatory domain joined by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the methylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-terminal methylesterase activity is stimulated, resulting in the subsequent demethylation of the chemoreceptors. We have investigated the mechanism of regulation of CheB activity by the N-terminal regulatory domain. First, we have found that phosphorylation of the N-terminal domain not only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutations in CheB that show an enhancement of methylesterase activity in the absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector domains. On the basis of these observations, we propose a model for activation of CheB in which phosphorylation of the regulatory domain results in a reorganization of the domain interface, allowing exposure of the active site to the receptor substrate and simultaneously stimulating methylesterase activity.

Bacterial Proteins↗
PubMed1998

Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain.

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein-protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

Bacterial Proteins↗
PubMed1998

Signal transduction in bacteria: molecular mechanisms of stimulus-response coupling.

In bacteria, adaptive responses to changing environmental conditions are mediated by signal transduction systems that involve modular protein domains. Despite great diversity in the integration of domains into different systems, studies of individual components have revealed molecular strategies that are widely applicable. Studies of receptors have advanced our understanding of how information is transmitted across membranes, the determination of three-dimensional structures of domains of histidine protein kinase domains and response regulator proteins has begun to reveal the molecular basis of signaling via two-component phosphoryltransfer pathways, and the description of 'eukaryotic-like' protein domains involved in bacterial signaling has emphasized the universality of intracellular signaling mechanisms.

Bacteria↗
PubMed1998

Chemotaxis receptor recognition by protein methyltransferase CheR.

Signal transduction processes commonly involve reversible covalent modifications of receptors. Bacterial chemotaxis receptors are reversibly methylated at specific glutamate residues within coiled-coil regions of their cytoplasmic domains. Methylation is catalyzed by an S-adenosylmethionine-dependent protein methyltransferase, CheR, that binds to a specific sequence at the C-termini of some chemotaxis receptors. From this tethering point, CheR methylates neighboring receptor molecules. We report the crystal structure, determined to 2.2 A resolution, of a complex of the Salmonella typhimurium methyltransferase CheR bound to the methylation reaction product, S-adenosylhomocysteine (AdoHcy), and the C-terminal pentapeptide of the aspartate receptor, Tar. The structure indicates the basis for the specificity of interaction between the chemoreceptors and CheR and identifies a specific receptor binding motif incorporated in the CheR methyltransferase domain.

Amino Acid Sequence↗