Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation

Abstract

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 (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO 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 TarFO 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 TarFO 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 TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.

A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species

“…Similarly, Td and Tm receptors belong to the same evolutionary class (44H [3]) as Bs receptors, whereas Ec receptors belong to another (36H). Furthermore, the unmodified receptors from Td and Tm deactivate CheA, which is a behavior consistent with an attractant-activating system like Bs (26,34). Indeed, these considerations and data presented here support a model wherein Td/Tm CheA activity decreases with repellent (O 2 ) and increases with attractant (Fe[II]).…”

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain

“…Atomic coordinates for E. coli CheA.P3. P4.P5 and CheW were derived from existing T. maritima crystal structures 57,58 using template-based homology modelling. An all-atom model of the E. coli Tsr (residues 1-518) was constructed from existing crystal structures of the ligand binding and cytoplasmic domains as well as a HAMP homology model based on a crystal structure of A. fulgidus HAMP.…”

Cooperation of two distinct coupling proteins creates chemosensory network connections

“…In other systems, CheA becomes substantially more active when coupled to a chemoreceptor (26,27). We thus incubated purified CheA with TlpD and either CheV1 or CheW (Fig.…”