Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems

Abstract

Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins.

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

“…The phosphotransfer kinetics support the hypothesis that transmembrane signal transduction is almost instantaneous, involving low affinity, transient AgrC-AgrA interactions. The rapid phosphotransfer is also consistent with a recent study that suggests that rapid phosphotransfer rates can be correlated with histidine kinase response regulator specificity and governs fidelity in twocomponent systems (32,33). As hypothesized by the molecular model, these interactions involve only the receiver domain of AgrA.…”

“…The intensities were plotted against time, allowing the rates of phosphorylation to be calculated as initial rates from the progress curves. The amount of phosphorylated AgrC Cyto (AgrC Cyto ϳP) was quantified by using a standard curve generated with known concentrations of [␥- 32 2 ). The reaction mixture was incubated at 25°C, and samples (12 l) were removed at different times, quenched with 3 l of 5ϫ SDS sample buffer, and visualized by 15% SDS-PAGE.…”

Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

“…HsfB and HsfA form a classical two-component system (37); however, it is not known what activates this pathway or if HsfB is capable of cross talk to another response regulator. This result is of great interest, as M. xanthus is known for its large repertoire of two-component systems regulating its complex lifestyle (36)(37)(38). It is also important to point out that the hsf cluster encodes a putative GGDEF-containing protein, suggesting that local concentrations of the second messenger, cyclic di-GMP (c-di-GMP), may affect predation.…”

“…S4 in the supplemental material) and includes genes encoding HsfB, HsfA (NtrC-like response regulator), an NAD kinase, and a protein with a conserved domain of unknown function (DUF218). In previous work, we demonstrated specific phosphotransfer between HsfB and HsfA (37). In other previous work, HsfA was shown to affect Lipoprotein, encoded in operon with two sensor box histidine kinases 1ϫ LOF the production of specialized metabolites, including DKxanthene and myxovirescin.…”

Chemosensory Regulation of a HEAT-Repeat Protein Couples Aggregation and Sporulation in Myxococcus xanthus

“…CheY7, CheY7 D53A , CheY7 F107A , and Cpc7 were cloned into pET28a expression vector to generate a T7-6ϫ His N-terminal tag. For acetyl phosphate (AcP) experiments, proteins were purified by standard batch procedure as previously described (23). For fluorescence binding assays, proteins were affinity purified over a Ni 2ϩ column and a size exclusion column.…”