Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.

binary enzyme-substrate (2; throughout the text, the bold numbers refer to species in Fig. The Catalytic Pathway of 1) and enzyme-product (4) complexes have Cytochrome P450cam a t been so characterized (4). Some features of the dioxygen-bound or activated oxygen intermediates, in particular the geometry of the Atomic Resolution six-coordinate low-spin heme, have been deduced from the structure of the ferrous llme ~chlichting,'* Joel Berendzen,' Kelvin Chu,'? Ann M. S t~c k ,~ (FelI+) carbonmonoxy complex (3) of Shelley A. M a v e~,~ David E. enso on,^ Robert M. S~e e t ,~ P450cam (5).However, the binding of carbon Dagmar Ringe,6 Gregory A. ~e t s k o ,~ monoxide to heme is likely to be different in Stephen G. Sligar'~~ a number of important ways from the binding Members o f t h e cytochrome P450 superfamily catalyze t h e addition o f m o -of oxygen ( 6 ) , and regardless, carbon monlecular oxygen t o nonactivated hydrocarbons a t physiological temperature-a oxide is an inhibitor, not a substrate, of reaction t h a t requires high temperature t o proceed i n t h e absence o f a catalyst. P450cam. Hence, the primary evidence for Structures were obtained for three intermediates i n the hydroxylation reaction the structures of the ferrous enzyme-substrate o f camphor b y P450cam w i t h trapping techniques and cryocrystallography. The complex (5), the dioxy intermediate (6), and structure o f t h e ferrous dioxygen adduct o f P450cam was determined w i t h 0.91

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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.

The molecular mechanisms responsible for stimulus-response coupling often involve two types of enzymatic components: histidine protein kinases (HPK), and their associated response regulators (RR). Signal transduction occurs through the transfer of phosphoryl groups from adenosine triphosphate (ATP) to histidine residues in the histidine kinases, from the HPK-phosphohistidine side chains to aspartic acid residues in the RR, and, finally, from the response regulator-phosphoaspartate side chains to water:

When the dynamic properties of many different proteins are plotted as a function of temperature, biphasic behaviour is observed, with a broad transition centred around 220 K. Atomic mean-square displacements from X-ray crystallography and Mössbauer scattering show this behaviour, as do electron transfer rates and dynamic information from inelastic neutron scattering. Molecular dynamics simulations over a range of temperatures also exhibit a transition at about 220 K: high-temperature atomic fluctuations are dominated by anharmonic collective motions of bonded and nonbonded groups of atoms, but below 220 K the predominant dynamic behaviour is harmonic vibration of individual atoms. Here we show by high-resolution X-ray diffraction that crystalline ribonuclease A does not bind substrate or inhibitor at 212 K but will bind either rapidly at 228 K. Once bound at the higher temperature, inhibitor cannot be washed off after the enzyme is cooled to below the transition temperature. These results suggest that enzyme flexibility is required for catalytic function.

Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that' a residue that is conserved in all homologous response regulator proteins (5). Two additional highly conserved residues, Asp-12 and Asp-13, bind a Mg2+ ion that is essential for phosphorylation (6, 7). CheY, like many other response regulators, has an associated autophosphatase activity; phospho-CheY has a half-life of =10 sec. Phosphatase activity is enhanced by an auxiliary regulatory protein, CheZ (8).The activity of response regulators is controlled by a family of histidine kinases that are autophosphorylated in the presence of ATP (1-3). The phosphotransfer mechanism involves the intermediate formation of a phosphohistidine residue in the kinase. In chemotaxis, the rate of phosphorylation of the histidine residue in the kinase, CheA, is stimulated by membrane chemoreceptor proteins (9, 10). The phosphoryl group is then rapidly transferred to CheY to control motility. Another chemotaxis response regulator, CheB, also accepts phosphoryl groups from CheA. -CheB provides a feedback adaptation mechanism. Phosphorylation of its N-terminal regulatory domain stimulates a C-terminal catalytic activity that modifies the chemoreceptors to attenuate CheA kinase activity (9, 11).Although in many cases a specific kinase has been implicated in the regulation of a given response regulator, considerable cross specificity has also been observed (12-14). The phenotypes of kinase mutants have indicated that response regulators can be phosphorylated in the absence of their cognate kinases (15-20). Here we show that CheY and CheB are enzymes that catalyze their own phosphorylation using low molecular weight phospho-donors. Thus, the enzymology of aspartate phosphorylation is an inherent property of the response regulators that can occur independently of any other protein. Proteins. Wild-type and mutant CheY proteins as well as CheZ and CheB were purified as described (23-25). Protein purity was estimated to be >95% on the basis of SDS/PAGE. MATERIALS AND METHODS Materials[3H]Methyl-labeled Tar receptor in Escherichia coli membranes was prepared as described (25 718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Cells display a remarkable ability to respond to small fluctuations in their surroundings. In simple microbial systems, information from sensory receptors feeds into a circuitry of regulatory proteins that transfer high energy phosphoryl groups from histidine to aspartate side chains. This phosphotransfer network couples environmental signals to an array of response elements that control cell motility and regulate gene expression.