Two-component signal transduction based on phosphotransfer from a histidine protein kinase to a response regulator protein is a prevalent strategy for coupling environmental stimuli to adaptive responses in bacteria. In both histidine kinases and response regulators, modular domains with conserved structures and biochemical activities adopt different conformational states in the presence of stimuli or upon phosphorylation, enabling a diverse array of regulatory mechanisms based on inhibitory and/or activating protein-protein interactions imparted by different domain arrangements. This review summarizes some of the recent structural work that has provided insight to the functioning of bacterial histidine kinases and response regulators. Particular emphasis is placed on identifying features that are expected to be conserved among different two-component proteins from those that are expected to differ, with the goal of defining the extent to which knowledge of previously characterized two-component proteins can be applied to newly discovered systems.

Response regulators (RRs) comprise a major family of signaling proteins in prokaryotes. A modular architecture that consists of a conserved receiver domain and a variable effector domain enables RRs to function as phosphorylation-regulated switches that couple a wide variety of cellular behaviors to environmental cues. Recently, advances have been made in understanding RR functions both at genome-wide and molecular levels. Global techniques have been developed to analyze RR input and output, expanding the scope of characterization of these versatile components. Meanwhile, structural studies have revealed that, despite common structures and mechanisms of function within individual domains, a range of interactions between receiver and effector domains confer great diversity in regulatory strategies, optimizing individual RRs for the specific regulatory needs of different signaling systems.

SummaryResponse regulator proteins exploit different molecular surfaces in their inactive and active conformations for a variety of regulatory intra-and/or intermolecular protein-protein interactions that either inhibit or activate effector domain activities. This versatile strategy enables numerous regulatory mechanisms among response regulators. The recent accumulation of structures of inactive and active forms of multi-domain response regulators and response regulator complexes has revealed many different domain arrangements that have provided insight into regulatory mechanisms. Although diversity is the rule, even among subfamily members containing homologous domains, several structural modes of interaction and mechanisms of regulation recur frequently. These themes involve interactions at the α4-β5-α5 face of the receiver domain, modes of dimerization of receiver domains, and inhibitory or activating hetero-domain interactions.

Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal transduction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simultaneous actions of multiple TCS activities. Here, we report a strategy that combines concentration-dependent phosphorylation profiling and mathematical modeling to characterize the cellular activities of the archetype Escherichia coli PhoR/PhoB system. Phosphorylation of the response regulator (RR) PhoB has been found to be dependent on the total concentrations of PhoB/PhoR and saturated at high concentrations. The relationship between RR phosphorylation and total concentrations has been defined by the modeling of the kinase and phosphatase reactions and quantified to derive the biochemical parameters of the PhoR/PhoB system in vivo. In a further test of this approach on a PhoB mutant, PhoB , it proved highly effective in exploring the mechanistic differences of TCSs that are not revealed by traditional reporter assays. Measurement of biochemical parameters for PhoB F20D led to the discovery that a weaker interaction between the histidine sensor kinase and RR could result in a higher and nonrobust phosphorylation due to diminished phosphatase activities.in vivo phosphorylation | two-component signal transduction T wo-component systems (TCSs), one of the predominant signaling schemes in bacteria, connect input stimuli and output responses with a core phosphotransfer between a histidine sensor kinase (HSK) and a cognate response regulator (RR) (1, 2). The signaling pathway is often simply described as a series of steps that include autophosphorylation of HSKs, phosphotransfer to cognate RRs, and output modulation, usually via transcription regulation, mediated by phosphorylated RRs. Far from a simple on/off switch, phosphorylation levels of many TCS proteins are under sophisticated control by multiple enzymatic activities. One of the fundamental questions in TCS studies is what percentage of protein molecules are phosphorylated in the presence or absence of the stimuli, but the exact phosphorylation levels have not been well quantified in vivo. Without the quantification of phosphorylation, it is extremely difficult to characterize TCS kinase and phosphatase activities in their native cellular environments. This has been identified as one of the key questions outstanding in TCS research (3, 4).RR phosphorylation levels are commonly inferred from transcriptional reporter activities even though gene transcription is downstream of RR phosphorylation and often complicated by additional regulatory factors. A change in transcription could result from alteration of RR phosphorylation levels as well as effects of additional uniden...

Response regulators function as the output components of two-component systems, which couple the sensing of environmental stimuli to adaptive responses. Response regulators typically contain conserved receiver (REC) domains that function as phosphorylation-regulated switches to control the activities of effector domains that elicit output responses. This modular design is extremely versatile, enabling different regulatory strategies tuned to the needs of individual signaling systems. This review summarizes structural features that underlie response regulator function. An abundance of atomic resolution structures and complementary biochemical data have defined the mechanisms for response regulator enzymatic activities, revealed trends in regulatory strategies utilized by response regulators of different subfamilies, and provided insights into interactions of response regulators with their cognate histidine kinases. Among the hundreds of thousands of response regulators identified, variations abound. This article provides a framework for understanding structural features that enable function of canonical response regulators and a basis for distinguishing noncanonical configurations.