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J A Hoch

Publications and source records attributed to J A Hoch.

At least 19 recordsLinked to original sources
PubMed2004

Developing inhibitors to selectively target two-component and phosphorelay signal transduction systems of pathogenic microorganisms.

Two-component signal transduction systems and their expanded variants known as phosphorelays are integral elements of the virulence and antimicrobial resistance responses of a wide range of pathogenic bacteria and fungi and also regulate essential functions. As a consequence, two-component systems and phosphorelays are recognized targets for the development of novel antimicrobial agents and a number of chemically synthesized inhibitors from different chemical classes have been identified by compound library screens. However, in the majority of cases these compounds do not appear to be selective for signal transduction pathways and exert their effect by multiple mechanisms of action. The key to designing molecules to selectively disrupt signal transduction may lie with the conserved features of response regulators and the structural analysis of complexes of signaling proteins.

Anti-Infective Agents↗
PubMed2002

Histidine kinase-mediated signal transduction systems of pathogenic microorganisms as targets for therapeutic intervention.

Pathogenic bacteria must be able to sense and respond rapidly to signals emanating from the host environment and use the signals to modulate the expression of genes required for the infection process. Two-component signal transduction systems, and their more complex variants known as phosphorelays, are woven within the fabric of bacterial cellular regulatory processes and are used to regulate the expression of genes involved in the virulence and antibiotic resistance responses of a large number of pathogens of major public health concern. The emergence of strains of pathogenic bacteria that are resistant to multiple antibiotics has driven the search for new targets and/or modes of action for anti-microbial agents. The presence of essential two-component systems in bacteria and the central role that these regulatory systems play in virulence and antibiotic resistance has meant that two-component systems and phosphorelays have been recognized as targets for antimicrobial intervention. This review will discuss the role of these signal transduction pathways in virulence responses and antibiotic sensitivity of pathogenic microorganisms and their potential use as targets for antimicrobial therapy. In addition, the current status on the development of inhibitors specific for two-component systems will be discussed.

Amino Acid Sequence↗
PubMed2001

PAS-A domain of phosphorelay sensor kinase A: a catalytic ATP-binding domain involved in the initiation of development in Bacillus subtilis.

The major sensor kinase controlling the initiation of development in Bacillus subtilis, KinA, functions by activating the phosphorelay signal-transduction system in response to as yet unknown signal ligands. KinA contains, within its amino-terminal signal-sensing region, three PAS domains that, in other proteins, are known to be involved in sensing changes in oxygen concentration and redox potential among other functions. The most amino-terminal PAS domain, PAS-A, was found to bind ATP and catalyze exchange of phosphate between ATP and nucleoside diphosphates. A cysteine-to-alanine mutation in PAS-A increased the affinity for ATP 5-fold, decreased the exchange reaction 2-fold, and stimulated KinA-dependent sporulation. A model for the role of ATP and the exchange reaction in the PAS domain in sensor kinase signal transduction is presented in which the free energy of nucleotide hydrolysis drives the conformational changes that activate or deactivate the sensor kinase in response to signal ligand binding.

Adenosine Triphosphate↗
PubMed2001

Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

Adenosine Triphosphate↗
PubMed2000

The mechanism of action of inhibitors of bacterial two-component signal transduction systems.

Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs.

Anti-Bacterial Agents↗
PubMed2000

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Bacillus subtilis↗
PubMed2000

Two-component and phosphorelay signal transduction.

Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Recent results have implicated these systems in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and developmental pathways. Elucidation of the structures of the interacting domains is leading to an understanding of the mechanisms of molecular recognition and phosphotransfer in these systems.

Bacteria↗
PubMed2000

Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis.

Protein homology studies identified five kinases potentially capable of phosphorylating the Spo0F response regulator and initiating sporulation in Bacillus subtilis. Two of these kinases, KinA and KinB, were known from previous studies to be responsible for sporulation in laboratory media. In vivo studies of the activity of four of the kinases, KinA, KinC, KinD (ykvD) and KinE (ykrQ), using abrB transcription as an indicator of Spo0A approximately P level, revealed that KinC and KinD were responsible for Spo0A approximately P production during the exponential phase of growth in the absence of KinA and KinB. In vitro, all four kinases dephosphorylated Spo0F approximately P with the production of ATP at approximately the same rate, indicating that they possess approximately equal affinity for Spo0F. All the kinases were expressed during growth and early stationary phase, suggesting that the differential activity observed in growth and sporulation results from differential activation by signal ligands unique to each kinase.

Bacillus subtilis↗
PubMed1999

Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation.

Five single alanine substitution mutations in the Spo0F response regulator gave rise to mutant strains of Bacillus subtilis with seemingly normal sporulation that nevertheless rapidly segregated variants blocked in sporulation. The basis for this deregulated phenotype was postulated to be increased phosphorylation of the Spo0A transcription factor, resulting from enhanced phosphate input or decreased dephosphorylation of the phosphorelay. Strains bearing two of these Spo0F mutant proteins, Y13A and I17A, retained a requirement for KinA and KinB kinases in sporulation, whereas the remaining three, L66A, I90A and H101A, gave strains that sporulated well in the absence of both KinA and KinB. Sporulation of strains bearing L66A and H101A mutations was decreased in a mutant lacking KinA, KinB and KinC, but the strain bearing the I90A mutation required the further deletion of KinD to lower its sporulation frequency. The affected residues, L-66, I-90 and H-101, are involved in crucial hydrophobic contacts stabilizing the orientation of helix alpha4 of Spo0F. The data are consistent with the notion that these three mutations alter the conformation of the beta4-alpha4 loop of Spo0F that is known to contain residues critical for KinA:Spo0F recognition. As this loop has a propensity for multiple conformations, the spatial arrangement of this loop may play a critical role in kinase selection by Spo0F and might be altered by regulatory molecules interacting with Spo0F.

Alanine↗
PubMed1999

ScoC regulates peptide transport and sporulation initiation in Bacillus subtilis.

Oligopeptides are transported into Bacillus subtilis by two ABC transport systems, App and Opp. Transcription of the operon encoding the Opp system was found to occur during exponential growth, whereas the app operon was induced at the onset of stationary phase. Transcription of both operons was completely curtailed by overproduction of the ScoC regulator from a multicopy plasmid and was enhanced in strains with the scoC locus deleted. ScoC, a member of the MarR family of transcription regulators, is known from previous studies to be a negative regulator of sporulation and of protease production that acts by binding directly to the promoters of the genes it regulates. Since peptide transport is essential for inactivation of the negative regulation of sporulation by Rap phosphatases, the control of ScoC transcription repression activity plays a crucial role in the initiation of sporulation.

ATP-Binding Cassette Transporters↗
PubMed1998

Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB.

The phosphorelay signal transduction pathway controls sporulation initiation in Bacillus subtilis. Transfer of a phosphoryl group from multiple kinases (KinA and KinB) through a single domain response regulator homologue (Spo0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regulator, (Spo0A) activates sporulation. Counteracting this response are phosphatases (RapA and RapB), which can short-circuit this phosphorelay via dephosphorylation of Spo0F. In vitro assays of RapB activity on phosphorylated Spo0F alanine-scanning mutants have been used to identify Spo0F residues critical for interactions between these proteins. The Spo0F surface comprised of the beta1-alpha1 loop and N-terminal half of helix alpha1 has the largest number of residues in which an alanine substitution leads to resistance or decreased sensitivity to RapB phosphatase activity. Other mutations desensitizing Spo0F to RapB are also located near the site of phosphorylation on the beta3-alpha3 and beta4-alpha4 loops. This surface is similar to but not the same as the surface identified for KinA and Spo0B interactions with Spo0F. Divalent metal ions were shown to be required for RapB activity, and this activity was insensitive to vanadate, suggesting that Rap phosphatases catalyze acyl phosphate hydrolysis by inducing conformational changes in phosphorylated Spo0F, which results in increased autodephosphorylation. Arginine 16 of Spo0F is proposed to play a role in catalysis, and similarities between the mechanisms for RapB catalyzed Spo0F approximately P hydrolysis and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are discussed.

Alanine↗
PubMed1998

Phosphorylation of the Spo0B response regulator phosphotransferase of the phosphorelay initiating development in Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay, a complex signal transduction system consisting of kinases and response regulators. The key component of a phosphorelay is the phosphotransferase, which recognizes two response regulators and transfers a phosphoryl group between them. In this reaction, the phosphoryl of one response regulator is transferred to a histidine on the phosphotransferase before phosphorylating an aspartate of the second response regulator. The phosphorylated histidine on the Spo0B phosphotransferase was found to be His30. Site-directed mutation of His30 to alanine destroyed its phosphotransferase activity in vitro and strains constructed with this mutation were unable to sporulate. None of the other 10 histidines of Spo0B was implicated in the phosphotransferase reaction. A structurally vulnerable site, histidine 23, was also identified through the mutational study. The His30 of Spo0B resides in a domain with little sequence homology to functionally equivalent domains in the phosphorelays of other bacterial and yeast systems, suggesting that the two types of phosphotransfer domains evolved convergently.

Amino Acid Sequence↗
PubMed1998

A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site.

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.

Adenosine Triphosphate↗
PubMed1998

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

Anti-Bacterial Agents↗