Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism.

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'-(beta,gamma-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 A) 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.

Comparison of the Pseudomonas aeruginosa and Escherichia coli PhoQ sensor domains: evidence for distinct mechanisms of signal detection.

The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.

Signal detection by the PhoQ sensor-transmitter. Characterization of the sensor domain and a response-impaired mutant that identifies ligand-binding determinants.

The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria. The PhoQ protein responds to low concentrations of extracellular divalent cations by activating PhoP-mediated transcription of a set of genes. PhoQ is a member of a family of transmembrane proteins that contain a periplasmic sensor domain coupled to a cytoplasmic transmitter domain. Here, we describe the cloning, purification, and properties of a fragment of Escherichia coli PhoQ corresponding to the sensor domain. This fragment is monomeric in solution and has a circular dichroism spectrum indicative of a mixture of alphahelix and beta-sheet. Divalent cations do not affect the oligomeric state, circular dichroism spectrum, or fluorescence spectrum of the sensor domain but do stabilize this domain to denaturation in a fashion expected for a direct binding model. We have also constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ). The mutant sensor domain is indistinguishable from wild type in terms of oligomeric form and spectral properties but differs in being substantially more stable to urea denaturation, showing no additional stabilization in the presence of divalent cations, and showing little activation of PhoP-mediated transcription in response to divalent-cation starvation in vivo. These data are consistent with a model in which divalent cations bind to the acidic cluster of the wild-type sensor domain and stabilize a conformation that is inactive in signaling. Substituting uncharged residues for the acidic cluster appears to mimic the effect of divalent-cation binding by stabilizing the inactive conformation.

Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure.

In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.

Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates.

Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Arc dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Arc variants labeled with fluorescence acceptor or donor groups and used energy transfer to monitor the increase in heterodimer with time. Under the conditions of this experiment, the rate at which the heterodimer concentration approaches its equilibrium value is determined by rate of dissociation and unfolding of the protein. Using this method and traditional denaturant-jump experiments, rate constants for unfolding/dissociation were determined over a wide range of stabilizing and destabilizing conditions. In each case examined, plots of log(ku) versus denaturant showed significant curvature under strongly denaturing conditions, even though other kinetic experiments indicates that the unfolding/dissociation reactions remain largely two-state. This curvature can be explained most readily by a series of unstable intermediates in the unfolding pathway, with denaturant-induced changes in the kinetic step that is rate-limiting. Alternatively, curvature might result from Hammond behavior in which the structure of the transition state becomes more native-like as the stability of native Arc decreases with increasing denaturant.

Sequence determinants of folding and stability for the P22 Arc repressor dimer.

The Arc repressor is a small, homodimeric protein. Studies of mutant proteins show that the side chains that form the hydrophobic core are the most important determinants of structure. A variety of hydrogen bonds and salt bridges also contribute to stabilization of the native structure, but these can often be replaced by hydrophobic interactions. The transition state for folding/unfolding is dimeric and contains a large amount of buried hydrophobic surface, but the beta-sheet of native Arc is not formed. Moreover, relatively little side chain information appears to be used in the transition state, suggesting that tight packing of the hydrophobic core and optimization of hydrogen-bond geometry are events that occur later in folding.

Domains of Mnt repressor: roles in tetramer formation, protein stability, and operator DNA binding.

The Mnt repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of gene regulatory proteins. Proteolytic cleavage of Mnt with chymotrypsin reveals that it consists of two structural domains. Both domains are required for high-affinity operator binding. The N domain (residues 1-51) is dimeric and binds weakly but specifically to operator DNA. The C domain (residues 52-82) forms an independent alpha-helical, tetramerization domain and, by itself, has no DNA-binding activity. In intact Mnt, the N and C domains help to stabilize each other against denaturation but appear to be linked rather flexibly. Assays of the half-operator affinities of Mnt and the isolated N domain indicate that binding to adjacent half-sites in the whole operator is stabilized by protein-protein contacts between N domains in addition to protein-protein contacts between C domains.

P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding.

The kinetics of unfolding and refolding have been measured for a set of Arc repressor mutants bearing single amino acid substitutions at 44 of the 53 residue positions. Roughly half of the mutations cause significant changes in the unfolding and/or refolding rate constants. These substitutions alter the hydrophobic core, tertiary hydrogen bonds and salt bridges, and glycines with restricted backbone conformations. Overall, the mutations cause larger changes in the unfolding rates than the refolding rates, indicating that significantly less side-chain information is used between the denatured state and transition state than between the transition state and native state. The set of mutants displays reasonable Brønsted behavior, suggesting that many native interactions are partially formed in the transition state. Taken together, these observations suggest that the overall structure of most of the protein must be somewhat native-like in the transition state but without close, complementary packing of the hydrophobic core or good hydrogen bond geometry. Such a transition state is inconsistent with a model in which monomers fold to their correct conformations and then dock to form the dimer but supports a model in which folding and dimerization are concurrent processes.

Are buried salt bridges important for protein stability and conformational specificity?

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.