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...

In a functional screen of mammalian complementary DNA libraries, we identified moesin as a novel gene whose overexpression blocks infection by murine leukemia viruses and human immunodeficiency virus type 1 in human and rodent lines, before the initiation of reverse transcription. Knockdown of moesin by RNA interference resulted in enhanced infection, suggesting that even the endogenous basal levels of moesin in rat fibroblasts are sufficient to limit virus infection. Moesin acts as a crosslinker between plasma membrane and actin filaments, as well as a signal transducer in responses involving cytoskeletal remodeling. Moesin overexpression was found to downregulate the formation of stable microtubules, whereas knockdown of moesin increased stable microtubule formation. A virus-resistant mutant cell line also displayed decreased stable microtubule levels, and virus-sensitive revertants recovered from the mutant line showed restoration of the stable microtubules, suggesting that these cytoskeletal networks play an important role in early post-entry events in the retroviral lifecycle. Together, these results suggest that moesin negatively regulates stable microtubule networks and is a natural determinant of cellular sensitivity to retroviral infection.

Recombination between inverted repeats is RAD52 dependent, but reduced only modestly in the rad51Δ mutant. RAD59 is required for RAD51-independent inverted-repeat recombination, but no clear mechanism for how recombination occurs in the absence of RAD51 has emerged. Because Rad59 is thought to function as an accessory factor for the single-strand annealing activity of Rad52 one possible mechanism for spontaneous recombination could be by strand annealing between repeats at a stalled replication fork. Here we demonstrate the importance of the Rad52 single-strand annealing activity for generating recombinants by showing suppression of the rad52Δ, rad51Δ rad52Δ and rad52Δ rad59Δ inverted-repeat recombination defects by the rfa1-D228Y mutation. In addition, formation of recombinants in the rad51Δ mutant was sensitive to the distance between the inverted repeats, consistent with a replication-based mechanism. Deletion of RAD5 or RAD18, which are required for error-free post-replication repair, reduced the recombination rate in the rad59Δ mutant, but not in wild type. These data are consistent with RAD51-independent recombinants arising by a faulty template switch mechanism that is distinct from nascent strand template switching.

The PhoP-PhoQ two-component system plays a role in Mg 2؉ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg 2؉ but resulted in reduced sensitivity to extracellular Ca 2؉ , suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartylphosphate bond of phospho-PhoP.