Ann H. West

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.

The α-aminoadipate pathway for lysine biosynthesis in fungi

This review provides a description of the biochemistry and enzymology of the α-aminoadipate pathway for lysine biosynthesis in fungi. The α-aminoadipate pathway is unique to fungi and is thus a potential target for the rational design of antifungal drugs. The present state of knowledge of the mechanisms of the seven enzymes in the pathway is presented, as well as detailed information with respect to structures and mechanisms of homocitrate synthase, saccharopine reductase, and saccharopine dehydrogenase.

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.

Immunodominance of Antigenic Site B over Site A of Hemagglutinin of Recent H3N2 Influenza Viruses

H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A–E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006–07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008–09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). “Native ELISA” analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006–07 and/or 2008–09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.

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.

Fungal Skn7 Stress Responses and Their Relationship to Virulence

ABSTRACT The histidine kinase-based phosphorelay has emerged as a common strategy among bacteria, fungi, protozoa, and plants for triggering important stress responses and interpreting developmental cues in response to environmental as well as chemical, nutritional, and hormone signals. The absence of this type of signaling mechanism in animals makes the so-called “two-component” pathway an attractive target for development of antimicrobial agents. The best-studied eukaryotic example of a two-component pathway is the SLN1 pathway in Saccharomyces cerevisiae, which responds to turgor and other physical properties associated with the fungal cell wall. One of the two phosphoreceiver proteins known as response regulators in this pathway is Skn7, a highly conserved stress-responsive transcription factor with a subset of activities that are dependent on SLN1 pathway phosphorylation and another subset that are independent. Interest in Skn7as a determinant in fungal virulence stems primarily from its well-established role in the oxidative stress response; however, the involvement of Skn7 in maintenance of cell wall integrity may also be relevant. Since the cell wall is crucial for fungal survival, structural and biosynthetic proteins affecting wall composition and signaling pathways that respond to wall stress are likely to play key roles in virulence. Here we review the molecular and phenotypic characteristics of different fungal Skn7 proteins and consider how each of these properties may contribute to fungal virulence.

Crystal structures of ferrous horse heart myoglobin complexed with nitric oxide and nitrosoethane.

The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO‐containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua‐metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 Å. The Fe‐N‐O angle of 147° in hh MbNO is larger than the corresponding 112° angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352–356) but is similar to the 150° angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827–10836). The Fe‐N(O) bond length of 2.0 Å (this work) is longer than the 1.75 Å distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 Å from the imidazole Nϵ atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 Å. The nitroso O atom of the EtNO ligand is located 2.7 Å from the imidazole Nϵ atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein. Proteins 2003.

Functional studies of the Ssk1p response regulator protein of Candida albicans as determined by phenotypic analysis of receiver domain point mutants

The Candida albicans response regulator protein Ssk1p regulates oxidant adaptation through the MAPK HOG1 pathway. Deletion mutants lacking SSK1 are oxidant sensitive in vitro and are killed more than wild‐type (WT) cells by human neutrophils. Furthermore, the mutants are avirulent in an invasive murine model, and unable to adhere to human esophageal cells. Transcriptional profiling has indicated that approximately 25% of all changes occur in genes encoding cell wall and stress adaptation functions. In this study, we have investigated the role of amino acid residues in the Ssk1p receiver (or regulatory) domain by constructing point mutants at positions D556 (putative site of protein phosphorylation) and D513 (putative role in divalent metal binding, phosphorylation and conformational switching). For each point mutant, their sensitivity to a variety of oxidant stress conditions was assessed and correlated with in vitro phosphorylation of each Ssk1p receiver domain, phosphorylation of the Hog1p MAP kinase, and translocation to the nucleus. We show that a D556N mutant is sensitive to 5 mM H2O2 or t‐butyl hydroperoxide, similar to a gene knock‐out ssk1 mutant, even though Hog1p is phosphorylated in the D556N mutant. To resolve this apparent paradox, we also demonstrate that Hog1p translocation to the nucleus in the D556N mutant is significantly reduced compared with WT cells (CAF2‐1). In a second point mutant, D513 was changed to a lysine residue (D513K). This mutant had WT levels of resistance to peroxide, but in comparison to WT cells and the D556N mutant, morphogenesis (yeast to hyphae transition) was inhibited in 10% serum or in M‐199 medium at 37°C. In the D513K point mutant, constitutive phosphorylation of Hog1p was observed, suggesting that a non‐conservative change (D513K) traps Ssk1p in an active conformation and therefore constitutive Hog1p phosphorylation. The inhibition of morphogenesis in D513K is related to a downregulation of the transcription factors of morphogenesis, EFG1 and CPH1. Another non‐conserved point mutant (D556R) was also constructed and phenotypically was like the D513K mutant. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of Hog1p occurs in vivo due to the inability of Ssk1p to be phosphorylated at least in the D513K mutant. We speculate that the non‐conservative changes described above in Ssk1p response regulator may cause conformational changes in the Ssk1p that account for phenotype differences compared with the D556N mutant that are also Hog‐independent.

Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways

ABSTRACT The histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the Saccharomyces cerevisiae multistep two-component signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both S. cerevisiae and in Cryptococcus neoformans. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in S. cerevisiae, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on S. cerevisiae Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.

Effects of osmolytes on the SLN1-YPD1-SSK1 phosphorelay system from Saccharomyces cerevisiae.

The multistep His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative, and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1, and two response regulator proteins, SSK1 and SKN7. Under nonosmotic stress conditions, the SLN1 sensor kinase is active, and phosphoryl groups are shuttled through YPD1 to SSK1, therefore maintaining the response regulator protein in a constitutively phosphorylated state. The cellular response to hyperosmotic stress involves rapid efflux of water and changes in intracellular ion and osmolyte concentration. In this study, we examined the individual and combined effects of NaCl and glycerol on phosphotransfer rates within the SLN1-YPD1-SSK1 phosphorelay. The results show that the combined effects of glycerol and NaCl on the phosphotransfer reaction rates are different from the individual effects of glycerol and NaCl. The combinatory effect is likely more representative of the in vivo changes that occur during hyperosmotic stress. In addition, the effect of osmolyte concentration on the half-life of the phosphorylated SSK1 receiver domain in the presence/absence of YPD1 was evaluated. Our findings demonstrate that increasing osmolyte concentrations negatively affect the YPD1 x SSK1-P interaction, thereby facilitating dephosphorylation of SSK1 and activating the HOG1 MAP kinase cascade. In contrast, at the highest osmolyte concentrations, reflective of the osmoadaptation phase of the signaling pathway, the kinetics of the phosphorelay favor production of SSK1-P and inhibition of the HOG1 pathway.