Dataset Information

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy.

ABSTRACT: Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.

SUBMITTER: Bhatnagar J

PROVIDER: S-EPMC3341552 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy.

Bhatnagar Jaya J Sircar Ria R Borbat Peter P PP Freed Jack H JH Crane Brian R BR

Biophysical journal 20120501 9

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with che ...[more]

PMID: 22824284

Similar Datasets

Project description:The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by approximately 20 degrees . In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.

Project description:The histidine kinase CheA plays a central role in signal integration, conversion, and amplification in the bacterial chemotaxis signal transduction pathway. The kinase activity is regulated in chemotaxis signaling complexes formed via the interactions among CheA's regulatory domain (P5), the coupling protein CheW, and transmembrane chemoreceptors. Despite recent advancements in the understanding of the architecture of the signaling complex, the molecular mechanism underlying this regulation remains elusive. An interdomain linker that connects the catalytic (P4) and regulatory domains of CheA may mediate regulatory signals from the P5-CheW-receptor interactions to the catalytic domain. To investigate whether this interdomain linker is capable of both activating and inhibiting CheA, we performed in vivo screens to search for P4-P5 linker mutations that result in different CheA autokinase activities. Several CheA variants were identified with kinase activities ranging from 30% to 670% of the activity of wild-type CheA. All of these CheA variants were defective in receptor-mediated kinase activation, indicating that the natural receptor-mediated signal transmission pathway was simultaneously affected by these mutations. The altered P4-P5 linkers were sufficient for making significant changes in the kinase activity even in the absence of the P5 domain. Therefore, the interdomain linker is an active module that has the ability to impose regulatory effects on the catalytic activity of the P4 domain. These results suggest that chemoreceptors may manipulate the conformation of the P4-P5 linker to achieve CheA regulation in the platform of the signaling complex.IMPORTANCE The molecular mechanism underlying kinase regulation in bacterial chemotaxis signaling complexes formed by the regulatory domain of the histidine kinase CheA, the coupling protein CheW, and chemoreceptors is still unknown. We isolated and characterized mutations in the interdomain linker that connects the catalytic and regulatory domains of CheA and found that the linker mutations resulted in different CheA autokinase activities in the absence and presence of the regulatory domain as well as a defect in receptor-mediated kinase activation. These results demonstrate that the interdomain linker is an active module that has the ability to impose regulatory effects on CheA activity. Chemoreceptors may manipulate the conformation of this interdomain linker to achieve CheA regulation in the platform of the signaling complex.

Dataset Information

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy.

Publications

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets