Structural and Mutational Analysis of the PhoQ Histidine Kinase Catalytic Domain

Abstract

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...

Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase

“…These interactions would not allow for GTP binding, which has been otherwise reported in other HKs (34). Further reinforcing this ATP-specificity profile, the nucleotide-binding pocket in DesK displays a conserved water network (35,36), strongly stabilizing the complex by complete satisfaction of the H-bonding potential of the protein and the adenine base moiety in the site (Fig. 1D).…”

Bacterial Two-Component Systems: Structures and Signaling Mechanisms

“…Residues from the G1 and G3 boxes are involved in binding the adenosine moiety; those from the N and G2 boxes contact both the adenosine moiety and the triphosphates-Mg 2+ moiety. The structure of E. coli PhoQ in complex with AMPPNP reveals detailed binding interactions of the conserved boxes with ATP [23]. In the G1 box, the conserved Asp has a direct H-bond to the amino group and a water-mediated H-bond to N1 of adenine.…”

“…In the absence of ATP, this loop is partially disordered in crystal structures. Even in the presence of ATP, the ATPlid shows high flexibility, indicated by high B-factors or in some cases is partially disordered in crystal structures [21][22][23][24] …”

Repression of Escherichia coli PhoP-PhoQ Signaling by Acetate Reveals a Regulatory Role for Acetyl Coenzyme A

“…pNL2 (30) is a low-copynumber pSC101-derived plasmid that carries the phoN-lacZ transcriptional fusion. pAED4QTR (32) is an expression vector for the PhoQ cytoplasmic domain in which a fragment of phoQ encoding the cytoplasmic domain (codons 219 to 486) was fused to an initiating ATG downstream of the T7 10 promoter and ribosome binding site of pAED4 (18).…”