Preformed Soluble Chemoreceptor Trimers That MimicCellular Assembly States and Activate CheA Autophosphorylation

摘要

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-Pformation with the Tar variants are low compared to those for autophosphorylationby free CheA, and net phosphotransfer from CheA to CheY does not increasecommensurately with CheA autophosphorylation. Thus, the Tar variantsfacilitate slow conversion to an active form of CheA that then undergoesstable autophosphorylation and is capable of subsequent phosphotransferto CheY. Free CheA is largely incapable of phosphorylation but containsa small active fraction. Addition of TarFO to CheA promotesa planar conformation of the regulatory domains consistent with arraymodels for the assembly state of the ternary complex and differentfrom that observed with a single inhibitory receptor. Introductionof TarFO into E. coli cells activatesendogenous CheA to produce increased clockwise flagellar rotation,with the effects increasing in the presence of the chemotaxis methylationsystem (CheB/CheR). Overall, the TarFO modules demonstratethat trimerized signaling tips self-associate, bind CheA and CheW,and facilitate conversion of CheA to an active conformation.