CheA Kinase of bacterial chemotaxis: chemical mapping of four essential docking sites

Abstract

The chemotaxis pathway of Escherichia coli and Salmonella typhimurium is the paradigm for the ubiquitous class of 2-component signaling pathways in prokaryotic organisms. Chemosensing begins with the binding of a chemical attractant to a transmembrane receptor on the cell surface. The resulting transmembrane signal regulates a cytoplasmic, multiprotein signaling complex that controls cellular swimming behavior by generating a diffusible phosphoprotein. The minimal functional unit of this signaling complex, termed the core complex, consists of the transmembrane receptor, the coupling protein CheW, and the histidine kinase CheA. Though the structures of individual components are largely known and the core complex can be functionally reconstituted, the architecture of the assembled core complex has remained elusive. To probe this architecture, the present study has utilized an enhanced version of the protein-interactions-by-cysteine-modification method (PICM-beta) to map out docking surfaces on CheA essential for kinase activity and for core complex assembly. The approach employed a library of 70 single, engineered cysteine residues, scattered uniformly over the surfaces of the five CheA domains in a cysteine-free CheA background. These surface Cys residues were further modified by the sulfhydryl-specific alkylating agent, 5-fluorescein-maleimide (5FM). The functional effects of individual Cys and 5FM-Cys surface modifications were measured by kinase assays of CheA activity in both the free and core complex-associated states, and by direct binding assays of CheA associations with CheW and the receptor. The results define (i) two mutual docking surfaces on the CheA substrate and catalytic domains essential for the association of these domains during autophosphorylation, (ii) a docking surface on the CheA regulatory domain essential for CheW binding, and (iii) a large docking surface encompassing regions of the CheA dimerization, catalytic, and regulatory domains proposed to bind the receptor. To test the generality of these findings, a CheA sequence alignment was analyzed, revealing that the newly identified docking surfaces are highly conserved among CheA homologues. These results strongly suggest that the same docking sites are widely utilized in prokaryotic sensory pathways. Finally, the results provide new structural constraints allowing the development of improved models for core complex architecture.

Figure 1

Summary of structural models from previous studies. (A) Model of the core complex consisting of the transmembrane receptor oligomer, CheA kinase, and CheW coupling protein. The receptors are shown as a trimer-of-dimers (49, 50) based on a composite structural model (reviewed in ref 6). HAMP is a conserved domain of unknown structure (–53), while Gly Hinge is also a conserved element (54). Homodimeric CheA and monomeric CheW are schematic. (B) Structural model of CheA showing one subunit in shades of green and the other in shades of gray. Black identifies the 70 positions of Cys substitutions used in this study. Yellow marks catalytic residues including H48, the site of phosphorylation on the P1 domain, and the ATP binding residues on the P4 domain. The composite CheA structure is assembled from S. typhimurium P1 domain (12), E. coli P2 domain (10), and T. maritima P3+P4+P5 domain fragment (11). Dashed lines indicate extended flexible linkers of undetermined structure that connect the domains.

Figure 2

Summary of the effects of modifications on receptor-stimulated CheA kinase activity in the reconstituted signaling complex. (A) Effect of cysteine modification on receptor-stimulated CheA activity relative to that of Cysless CheA. Values below the indicated threshold are operationally defined as perturbed. (B) Effect of 5-fluorescein-maleimide (5FM) attachment on receptor-stimulated CheA activity relative to that of Cysless CheA. (C) PICM parameter (see text) for each cysteine position. Again, values below the indicated threshold are operationally defined as perturbed. (In panels A and C, five above-threshold modifications were ambiguous because their error bars crossed the threshold: H401C, T436C, R332F1, A412F1, M469F1. All five were found to be nonperturbing in the pull-down assay for core complex binding (56), confirming their identification as nonperturbing modifications and validating the indicated thresholds.)

Figure 3

Summary of the effects of 5-fluorescein-maleimide (5FM) attachment on CheA binding to its docking partners. (A) Effects of 5FM attachment on relative incorporation of CheA into the core complex as defined by the binding parameter B_C (see text, eq 1). The horizontal line indicates operational threshold of strong complex affinity. (B) Effects of 5FM attachment on free CheA binding to CheW. Values shown are the binding affinity (K_A = 1/K_D), relative to that of Cysless CheA.

Figure 4

Spatial mapping of results, and working models for core complex architecture. (A) Experimental PICM parameters. Nonperturbing positions (white) lie on surfaces with no detected docking interactions. Perturbing positions assigned to the P1–P4 interaction faces (blue), to the CheW docking site (red), and to an essential surface proposed to bind the receptor (black) are also indicated. (B) Conserved surface residues, where the intensity of red is proportional to the degree of conservation (see Supporting Information). (C and D) Top views of panels A and B, respectively. (E and F) Two working models for core complex architecture. P1, CheW, and receptor are shown in light green, blue, and tan, respectively.