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Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers.

ABSTRACT: Residues E402 and R404 of the Escherichia coli serine chemoreceptor, Tsr, appear to form a salt bridge that spans the interfaces between neighboring dimers in the Tsr trimer of dimers, a key structural component of receptor core signaling complexes. To assess their functional roles, we constructed full sets of single amino acid replacement mutants at E402 and R404 and characterized their signaling behaviors with a suite of in vivo assays. Our results indicate that the E402 and R404 residues of Tsr play their most critical signaling roles at their inner locations near the trimer axis where they likely participate in stabilizing the trimer-of-dimer packing and the kinase-ON state of core signaling complexes. Mutant receptors with a variety of side-chain replacements still accessed both the ON and OFF signaling states, suggesting that core signaling complexes produce kinase activity over a range of receptor conformations and dynamic motions. Similarly, the kinase-OFF state may not be a discrete conformation but rather a range of structures outside the range of those suitable for kinase activation. Consistent with this idea, some structural lesions at both E402 and R404 produced signaling behaviors that are not compatible with discrete two-state models of core complex signaling states. Those lesions might stabilize intermediate receptor conformations along the OFF-ON energy landscape. Amino acid replacements produced different constellations of signaling defects at each residue, indicating that they play distinct structure-function roles. R404, but not E402, was critical for high signal cooperativity in the receptor array.

SUBMITTER: Lai RZ

PROVIDER: S-EPMC5370551 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers.

Lai Run-Zhi RZ Gosink Khoosheh K KK Parkinson John S JS

Journal of molecular biology 20170216 6

Residues E402 and R404 of the Escherichia coli serine chemoreceptor, Tsr, appear to form a salt bridge that spans the interfaces between neighboring dimers in the Tsr trimer of dimers, a key structural component of receptor core signaling complexes. To assess their functional roles, we constructed full sets of single amino acid replacement mutants at E402 and R404 and characterized their signaling behaviors with a suite of in vivo assays. Our results indicate that the E402 and R404 residues of T ...[more]

PMID: 28215934

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Project description:Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (Tar(FO)). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the Tar(FO) modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The Tar(FO) variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of Tar(FO) to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of Tar(FO) into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the Tar(FO) modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.

Dataset Information

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers.

Publications

Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers.

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