The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiledcoil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPbN at 1.9 Å resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.

The side chains of Arg 31, Glu 36 and Arg 40 in Arc repressor form a buried salt-bridge triad. The entire salt-bridge network can be replaced by hydrophobic residues in combinatorial randomization experiments resulting in active mutants that are significantly more stable than wild type. The crystal structure of one mutant reveals that the mutant side chains pack against each other in an otherwise wild-type fold. Thus, simple hydrophobic interactions provide more stabilizing energy than the buried salt bridge and confer comparable conformational specificity.

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PhoQ is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems common in prokaryotes and lower eukaryotes. In response to changes in environmental Mg 2؉ concentration, PhoQ regulates the level of phosphorylated PhoP, its cognate transcriptional response-regulator. The PhoQ cytoplasmic region comprises two independently folding domains: the histidine-containing phosphotransfer domain and the ATP-binding kinase domain. We have determined the structure of the kinase domain of Escherichia coli PhoQ complexed with the non-hydrolyzable ATP analog adenosine 5-(␤,␥-imino)triphosphate and Mg 2؉ . Nucleotide binding appears to be accompanied by conformational changes in the loop that surrounds the ATP analog (ATP-lid) and has implications for interactions with the substrate phosphotransfer domain. The high resolution (1.6 Å) structure reveals a detailed view of the nucleotide-binding site, allowing us to identify potential catalytic residues. Mutagenic analyses of these residues provide new insights into the catalytic mechanism of histidine phosphorylation in the histidine kinase family. Comparison with the active site of the related GHL ATPase family reveals differences that are proposed to account for the distinct functions of these proteins. Two-component signaling systems are used ubiquitously by prokaryotes and also by a number of lower eukaryotes to sense and respond to various environmental conditions. These systems consist of a histidine kinase that acts as the sensor of environmental stimuli and a response regulator that mediates the cellular response, generally at the level of transcriptional control (1). As with many signaling pathways, protein phosphorylation is used as a means to transmit information; however, unlike the majority of phosphoproteins found in higher eukaryotes, in which tyrosine, serine, or threonine serve as the substrate for phosphorylation, histidine kinases autophosphorylate a histidine residue from which the phosphoryl group is subsequently transferred to a conserved aspartate residue in the response regulator. The catalytic mechanism is reasonably well understood for aspartyl phosphorylation, while far less is known about the autokinase reaction. This lack of information is due in part to the relative scarcity of detailed structural information for the histidine kinases. Recently, structural information has become available for the CheA (2, 3) and EnvZ (4) histidine kinases. These structures reveal that the catalytic ATP-binding domain is an autonomously folding ␣/␤-sandwich that shares structural homology with a family of ATPases that include Hsp90, DNA gyrase B, and MutL (5). Although these structures provide some insight into function, they have not allowed the assignment of catalytic residues. Here we describe the 1.6-Å resolution crystal structure of the catalytic domain of the PhoQ histidine kinase complexed with an AMPPNP 1 nucleotide. PhoQ is a transmembrane histidine kinase that is involved in Mg 2ϩ homeostasis and/or pathogenesis of...

The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg 2؉ , and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wildtype Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50 and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, nonphysiological dimeric association.