During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric, histidine-kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and x-ray scattering studies of thermostable CheA from Thermotoga maritima find that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP nor ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains, or removal of one within a dimer, increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates inter-subunit (trans) versus intra-subunit (cis) autophosphorylation; with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans-directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and suggests that the ATP-lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.

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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.

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Chemotaxis is the signal transduction mechanism that allows bacteria to change direction in response to an external stimulus. Detection of an environmental stimulus by the receptors has a cascading effect leading to the flagellar motor changing direction. This results in the tumbling of the bacterium. The receptors form hexagonal lattices at the poles of cells. The histidine kinase CheA and coupling protein CheW are localized to the lattice to form the ternary complex. CheA, the principal enzyme in the chemotaxis signaling pathway, is a multi domain protein comprised of: P1‐histidine phosphotransfer, P2‐regulatory, P3‐dimerization, P4‐kinase, and P5‐regulatory. From previous studies it is known that the transfer of the γ‐phosphate of ATP occurs between the P1 and P4 domain, however how autophosphorylation activity is regulated is not completely understood. To comprehend this transient interaction, variants (ΔP2) of Thermotoga maritima CheA were generated. Small angle X‐ray scattering (SAXS) data has shown that spatial changes occur upon addition of an ATP analog. To determine relative activity of the ΔP2 variants or CheA domains radioisotope assays were used. Point mutations on the full length and ΔP2 CheA showed whether autophosphorylation occurs cis or trans in the dimer. Crosslinking between residues near the active sites in the P4 and P1 domains enabled isolation of the autophosphorylation transfer conformation. The crystal structure of P3P4 domains reveals a more active configuration of the P4 domain. Progress towards understanding how the domains interact to facilitate the transfer of the ATP γ‐phosphate is not clear and will be discussed.