Identification and Ligand-Binding Mechanism Analysis of a C4-Dicarboxylate Chemoreceptor from Agrobacterium tumefaciens

Methyl-accepting chemotaxis proteins (MCPs, or chemoreceptors) function as sensors and signal transmitters of the chemotaxis signaling system. As a soil-borne facultative phytopathogen, Agrobacterium tumefaciens (or fabrum) C58 encodes 20 MCPs, but only seven have been experimentally verified. Here, we characterized Atu0646, predicted as a transmembrane chemoreceptor with an sCache_2-type periplasmic ligand-binding domain (LBD0646). Differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) results confirmed LBD0646 directly binds L-malate and succinate. Gene deletion assays validated Atu0646 as the chemoreceptor for these two ligands in A. tumefaciens C58. The chemotactic attraction of A. tumefaciens toward two plant-derived C4-dicarboxylates provides reasonable support for the critical role of chemotaxis in rhizosphere colonization and susceptible wound site localization of this phytopathogen. AlphaFold structural modeling, molecular docking, and site-directed mutagenesis identified key binding residues: five substitutions (Y90A, H103A, K155A, R125Y, Y174K) resulted in loss of function due to the alteration of its LBD structure, two (R108Y, N151Y) attenuated it, and one (S119Y) enhanced its binding capacity to its cognate ligand, while also broadening its ligand spectrum to recognize Asp. Ligand-binding mechanism analysis of Atu0646 improves our understanding of the structure–function relationship of chemoreceptors and provides a feasible strategy for the rational protein engineering of chemoreceptors.