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J A Hoch

Publications and source records attributed to J A Hoch.

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

Identification of communication networks in Spo0F: a model for phosphorylation-induced conformational change and implications for activation of multiple domain bacterial response regulators.

Fundamental to understanding the mechanism by which phosphorylation activates bacterial signal transduction response regulator proteins is the identification of regions and residues that are responsible for the phosphorylation-induced conformational change. Here we review results from structural and protein dynamics investigations, and combine them with mutagenesis studies on the response regulator protein SpoOF to suggest a model in which a network of buried and surface residues link surface regions required for protein:protein interactions to the site of phosphorylation. The network described for SpoOF may provide pathways through which information is transmitted from the site of phosphorylation, propagating a conformational change many angstroms away. The general applicability of the communication network model for all bacterial response regulator proteins is discussed.

Bacterial Proteins↗
PubMed1998

Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

Adenosine Diphosphate↗
PubMed1998

Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

Amino Acid Sequence↗
PubMed1998

Initiation of bacterial development.

The discovery of a mitotic apparatus in bacteria has led to significant recent progress being made in understanding the regulatory connections between the cell cycle, chromosome segregation and the onset of developmental processes in sporulation. The control of developmental transcription by antagonism between protein kinase and protein phosphatase reached a new level of complexity with the discovery of peptide inhibitors of protein phosphatases that cycle between the interior and exterior cell surface as information messengers. New mechanisms of developmental regulation are being uncovered in a variety of microbial systems.

4-Butyrolactone↗
PubMed1998

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown. In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

Bacillus subtilis↗
PubMed1998

A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy.

A two-component signal transduction system encoded by the yycF and yycG genes is part of an operon containing three genes, yycH, yycI, and yycJ, with no known function and a gene, yycK, coding for an HtrA-like protease. This operon was transcribed during growth, and its transcription shut down as the cells approached stationary phase. This decreased transcription was not Spo0A dependent. The HtrA protease gene was separately controlled during sporulation from a sigmaG promoter. Studies using insertional inactivation plasmids revealed that neither yycF nor yycG could be inactivated, whereas the other genes were inactivated without loss of viability. A temperature-sensitive YycF response regulator mutant was isolated and shown to have an H215P mutation in a putative DNA-binding domain which is closely related to the OmpR family of response regulators. At the nonpermissive temperature, cultures of the mutant strain stopped growth within 30 min, and this was followed by a decrease in optical density. Microscopically, many of the cells appeared to retain their structure while being empty of their contents. The essential processes regulated by this two-component system remain unknown. A search of the genome databases revealed YycF, YycG, and YycJ homologues encoded by three linked genes in Streptococcus pyogenes. The high level of identity of these proteins (71% for YycF) suggests that this system may play a similar role in gram-positive pathogens.

Amino Acid Sequence↗
PubMed1997

A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis.

Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.

Amino Acid Substitution↗
PubMed1997

A novel histidine kinase inhibitor regulating development in Bacillus subtilis.

Kinase A is the sensor histidine kinase responsible for processing postexponential phase information and providing phosphate input to the phosphorelay that activates developmental transcription via phosphorylated Spo0A. A protein inhibitor, KipI, of kinase A was discovered encoded in an operon of genes of unknown function but regulated by the availability of fixed nitrogen. KipI is a potent inhibitor of the autophosphorylation reaction of kinase A but does not inhibit phosphate transfer to the Spo0F response regulator once kinase A is phosphorylated. KipI is an inhibitor of the catalytic domain of kinase A affecting the ATP/ADP reactions and not the phosphotransferase functions of this domain. The inhibitory activity of KipI is counteracted by the product of another gene in the operon, KipA. This protein may bind to KipI, preventing its function as an inhibitor of kinase A. KipI may be the first representative of a new class of signal transduction inhibitors that function by direct interaction with the catalytic domain of histidine kinases to counteract signals influencing the "sensor" domain of such kinases. This inhibitor represents yet another way by which the phosphorelay signal transduction system is affected by negative regulators under the control of metabolic, environmental, or cell cycle influences antithetical to the initiation of developmental transcription.

Bacillus subtilis↗
PubMed1997

Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis.

The phosphorelay, a signal transduction pathway composed of two-component regulatory proteins, mediates the initiation of sporulation in Bacillus subtilis. Environmental and physiological signals activate the autophosphorylation of histidine kinases, KinA and KinB, which transfer the phosphoryl group to Spo0F, a single domain homolog of the two-component response regulator. Phosphorylated Spo0F passes the phosphate to the final transcriptional regulator, Spo0A, through a phosphotransferase, Spo0B. Spo0F shares significant homology with other members of the response regulator family. It displays a (beta/alpha)5-barrel scaffold with the active site situated at the carboxyl end of the beta strands. The molecular recognition of Spo0F with its cognate proteins was investigated using a comprehensive strategy termed alanine-scanning mutagenesis. Of the total 124 residues, 79 in the region of helices and loops were individually changed to alanine using site-directed mutagenesis. The mutants with notable in vivo sporulation phenotypes were further examined in vitro to identify the corresponding effect in each protein-protein interaction. This study revealed that most, if not all, protein-protein interactions involve the residues in the vicinity of the active site. The surface-exposed residues critical for the interactions with KinA or Spo0B were identified. Surprisingly, although these interaction proteins are very different, they recognize subsets of residues comprising a common surface of Spo0F.

Alanine↗
PubMed1997

High-resolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: implications for phosphorylation and molecular recognition.

NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F. This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis. Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue). The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms. Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns. Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites. Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region. These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions. Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation. These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state.

Bacillus subtilis↗
PubMed1997

Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein, Spo0B, from Bacillus subtilis.

The phosphotransferase protein Spo0B, a component of the sporulation signal transduction system in Bacillus subtilis was expressed from the Escherichia coli strain BL21DE3. It was purified, crystallized, and 2.25 A data measured using the synchrotron source at the Stanford Linear Accelerator Center. The search for heavy atom derivatives is in progress.

Bacillus subtilis↗
PubMed1997

Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis.

Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals. Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp-like genes coding for homologues of the Escherichia coli leucine-responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT). SHMT catalyses interconversion of serine and glycine while transferring the resulting one-carbon unit into the C1 pool through methylene tetrahydrofolate. Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S-adenosyl-methionine (SAM). The results of experiments using L-ethionine suggest that SAM is involved in post-synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB. Truncated LrpA and LrpB peptides that have retained the DNA-binding domain but have lost the C-terminal half of the protein appear to act as repressors of glyA transcription and KinB-dependent sporulation. However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB. Our results indicate that lrpA-lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.

Amino Acid Sequence↗
PubMed1997

KapB is a lipoprotein required for KinB signal transduction and activation of the phosphorelay to sporulation in Bacillus subtilis.

KinB is one of the two major histidine kinases that provide phosphate input in the phosphorelay to produce SpoOA approximately P, the key transcription factor controlling the initiation of sporulation. A search for insertion mutants affected in activation of KinB-dependent sporulation led to the identification of the lgt locus encoding the lipoprotein glyceryltransferase required for the lipid modification of prolipoproteins before their cleavage and translocation across the cytoplasmic membrane. In parallel, a putative lipoprotein signal peptide cleavage site was detected in KapB, known to be strictly required for KinB-mediated sporulation and located downstream of KinB in a single transcription unit. Using PhoA peptide fusions, we have shown that KapB signal-peptide can direct active alkaline phosphatase to the outer surface of the cytoplasmic membrane in an LGT-dependent manner, strongly suggesting that KapB is a lipoprotein tethered to the outer face of the cytoplasmic membrane via a lipid anchor. As KapB proved to be dispensable for expression of the kinBkapB operon, a chimeric kinase was built consisting of KinA sensor domain fused to KinB kinase domain (KinA'-'B) to assess (i) the involvement of KapB in catalysis of the kinase reaction, and (ii) the ability of KinB to phosphorylate SpoOF in vitro. It was shown that KapB is dispensable for both in vivo and in vitro activation of the phosphorelay by the KinA'-'B chimera and that KinA'-'B phosphorylates SpoOF directly in vitro. Models for the role of KapB in regulating KinB activity are discussed.

Alkaline Phosphatase↗
PubMed1996

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of a Y13S mutant of Spo0F from Bacillus subtilis.

Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A. Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05. A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

Journal Article↗