Histidine kinases and response regulator proteins in two-component signaling systems.

Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating histidine protein kinases provide phosphoryl groups for response regulator proteins which, in turn, function as molecular switches that control diverse effector activities. Structural studies of proteins involved in two-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.

Novel role for an HPt domain in stabilizing the phosphorylated state of a response regulator domain.

Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. In Saccharomyces cerevisiae, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.

Functional roles of conserved amino acid residues surrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1.

The histidine-containing phosphotransfer (HPt) protein YPD1 is an osmoregulatory protein in yeast that facilitates phosphoryl transfer between the two response regulator domains associated with SLN1 and SSK1. Based on the crystal structure of YPD1 and the sequence alignment of YPD1 with other HPt domains, we site-specifically engineered and purified several YPD1 mutants in order to examine the role of conserved residues surrounding the phosphorylatable histidine (H64). Substitution of the positively charged residues K67 and R90 destabilized the phospho-imidazole linkage, whereas substitution of G68 apparently reduces accessibility of H64. These findings, together with the effect of other mutations, provide biochemical support of the proposed functional roles of conserved amino acid residues of HPt domains.

Conservation of structure and function among histidine-containing phosphotransfer (HPt) domains as revealed by the crystal structure of YPD1.

In Saccharomyces cerevisiae, the SLN1-YPD1-SSK1 phosphorelay system controls a downstream mitogen-activated protein (MAP) kinase in response to hyperosmotic stress. YPD1 functions as a phospho-histidine protein intermediate which is required for phosphoryl group transfer from the sensor kinase SLN1 to the response regulator SSK1. In addition, YPD1 mediates phosphoryl transfer from SLN1 to SKN7, the only other response regulator protein in yeast which plays a role in response to oxidative stress and cell wall biosynthesis. The X-ray structure of YPD1 was solved at a resolution of 2.7 A by conventional multiple isomorphous replacement with anomalous scattering. The tertiary structure of YPD1 consists of six alpha-helices and a short 310-helix. A four-helix bundle comprises the central core of the molecule and contains the histidine residue that is phosphorylated. Structure-based comparisons of YPD1 to other proteins having a similar function, such as the Escherichia coli ArcB histidine-containing phosphotransfer (HPt) domain and the P1 domain of the CheA kinase, revealed that the helical bundle and several structural features around the active-site histidine residue are conserved between the prokaryotic and eukaryotic kingdoms. Despite limited amino acid sequence homology among HPt domains, our analysis of YPD1 as a prototypical family member, indicates that these phosphotransfer domains are likely to share a similar fold and common features with regard to response regulator binding and mechanism for histidine-aspartate phosphoryl transfer.

Purification, crystallization and preliminary X-ray diffraction analysis of the yeast phosphorelay protein YPD1.

YPD1 is a yeast osmoregulatory protein that functions in a phosphorelay signal-transduction pathway. YPD1 has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals were obtained by hanging-drop vapor-diffusion using PEG 4000 as a precipitant. Preliminary X-ray diffraction analysis indicates that the crystals belong to tetragonal space group P43212 or P41212 with unit-cell dimensions a = b = 52.71, c = 244.02 A. X-ray data to 2.7 and 3.0 A have been collected from native crystals and a heavy-atom derivative, respectively. Positions for two Hg atoms have been located by analysis of difference Patterson maps.

Differential stabilities of phosphorylated response regulator domains reflect functional roles of the yeast osmoregulatory SLN1 and SSK1 proteins.

Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration in Escherichia coli. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E. coli. A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.

Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain.

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein-protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

Crystal structure of the catalytic domain of the chemotaxis receptor methylesterase, CheB.

Signaling activity of bacterial chemotaxis transmembrane receptors is modulated by reversible covalent modification of specific receptor glutamate residues. The level of receptor methylation results from the activities of a specific S-adenosylmethionine-dependent methyltransferase, CheR, and the CheB methylesterase, which catalyzes hydrolysis of receptor glutamine or methylglutamate side-chains to glutamic acid. The CheB methylesterase belongs to a large family of response regulator proteins in which N-terminal regulatory domains control the activities of C-terminal effector domains. The crystal structure of the catalytic domain of the Salmonella typhimurium CheB methylesterase has been determined at 1.75 A resolution. The domain has a modified, doubly wound alpha/beta fold in which one of the helices is replaced by an anti-parallel beta-hairpin. Previous biochemical and mutagenesis data, suggest that the methylester hydrolysis catalyzed by CheB proceeds through a mechanism involving a serine nucleophile. The methylesterase active site is tentatively identified as a cleft at the C-terminal edge of the beta-sheet containing residues Ser164, His190 and Asp286. The three-dimensional fold, and the arrangement of residues within the catalytic triad distinguishes the CheB methylesterase from any previously described serine protease or serine hydrolase.

Purification, crystallization, and preliminary X-ray diffraction analyses of the bacterial chemotaxis receptor modifying enzymes.

Bacterial chemotaxis receptor modifying enzymes from Salmonella typhimurium have been crystallized using microseeding techniques. The crystals of the S-adenosyl-L-methionine-dependent methyltransferase, CheR, belong to the monoclinic space group P21 with cell constants a = 55.1 A, b = 48.1 A, c = 63.1 A, beta = 112.3 degrees. The crystals of the catalytic domain of the methylesterase, CheB, belong to the trigonal space group P3(2)21 or P3(1)21 with unit cell dimensions of a = b = 63.4 A, c = 86.8 A. Both crystals contain one molecule per asymmetric unit and have calculated Matthews' volumes of 2.4 A3/Da.

Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis.

The response regulator protein of bacterial chemotaxis, CheY, is representative of a large family of signal transduction proteins that function as phosphorylation-activated switches to regulate the activities of associated effector domains. These regulators catalyze the metal ion-dependent phosphoryl transfer and dephosphorylation reactions that control the effector activities. The crystal structures of Salmonella typhimurium CheY with and without Mg2+ bound at the active site have been determined and refined at 1.8-A resolution. While the overall structures of metal-bound and metal-free CheY are similar, significant rearrangements occur within the active site involving the three most highly conserved residues of the response regulator family. Conservation of the cluster of carboxylate side chains at the active site of response regulator domains can be rationalized in terms of their role in coordinating the catalytically essential divalent metal ion. The Mg2+ coordination geometry provides insights to the mechanism of phosphoryl transfer.

Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein.

The processing enhancing protein of mitochondria (PEP) is an essential component that has been shown to participate in proteolytic removal of NH2-terminal signal peptides from precursor proteins imported into the mitochondrial matrix. Using a yeast strain bearing a PEP mutation that renders it temperature-sensitive, an approach of genetic suppression was taken in order to identify additional components that could be involved with protein import: high copy plasmids comprising a yeast genomic library were tested for ability to suppress the 37 degrees C growth defect. Two plasmids were isolated, pSMF1 and pSMF2, which suppressed the growth defect nearly as well as the cloned PEP gene itself. Sequence analysis of the rescuing genes predicted extremely hydrophobic proteins with sizes of 63 and 60 kDa, respectively. Remarkably, the predicted SMF1 and SMF2 products are 49% identical to each other overall. To test the requirement for SMF1 and SMF2, the chromosomal genes were disrupted. Individual disruption was without effect, but cells in which both genes were disrupted grew poorly. When mitochondria were prepared from the double disruption strain grown in a nonfermentable carbon source, they were morphologically normal but defective for translocation of radiolabeled precursor proteins. SMF1 protein was provisionally localized to the mitochondrial membranes using epitope tagging. We suggest that SMF1 and SMF2 are mitochondrial membrane proteins that influence PEP-dependent protein import, possibly at the step of protein translocation.

Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2.

The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol. 141, 213-222). They include the origin of replication (oriRK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions oriRK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.

Comparison of methods for identification of Giardia lamblia.

Stool specimens from children in daycare centers were screened for Giardia lamblia and intestinal amoebae by staining wet mounts with methylene blue and dilute Lugol's iodine. Merthiolate-iodine-formalin concentrations (MIFC) and permanent smears stained with Wheatley's trichrome method also were done. In addition, stools were preserved with polyvinyl alcohol (PVA) and 10% formalin and tested with trichrome and MIFC, respectively. The effectiveness of each method was based on a quantification scheme. Trichrome and MIFC were the best identification methods for cysts of G. lamblia. Trichrome was the superior method for identification of trophozoites. The other staining procedures were significantly less accurate. The use of preservatives did not improve recovery of G. lamblia compared with same morning processing of fresh stools. This study provides evidence that a permanent stain such as trichrome is an important tool for the diagnosis of G. lamblia and should be included in the processing of any diarrheal stool.

Comparative in vitro activities of ten antimicrobial agents against bacterial enteropathogens.

The in vitro susceptibilities of 50 strains of Salmonella spp., 80 strains of Shigella spp., and 50 enterotoxigenic Escherichia coli, 14 Yersinia enterocolitica, 6 Aeromonas hydrophila, 4 Plesiomonas shigelloides, 9 Vibrio parahaemolyticus, and 30 Campylobacter jejuni strains that were recently isolated from worldwide sources were determined for 10 antimicrobial agents. The antimicrobial agents tested included ampicillin, bicozamycin, doxycycline, enoxacin (CI-919), erythromycin, furazolidone, amdinocillin, norfloxacin, trimethoprim, and trimethoprim-sulfamethoxazole. Ampicillin resistance occurred frequently in strains of Salmonella and Shigella spp. and enterotoxigenic E. coli strains. The most active agents against all of the bacteria tested were enoxacin and norfloxacin. Furazolidone and amdinocillin were also highly active against the majority of strains. Trimethoprim and trimethoprim-sulfamethoxazole were inhibitory at low concentrations against all test except C. jejuni isolates. The in vitro results of this study confirm the high prevalence of bacterial resistance to ampicillin. However, this work also identifies four antimicrobial agents, enoxacin, furazolidone, norfloxacin, and amdinocillin, that would be appropriate for further testing in clinical trials.