Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.     

Nucleoside diphosphate kinase B knock-out mice have impaired activation of the K+ channel KCa3.1, resulting in defective T cell activation

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K(+) channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca(2+) flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B(-/-) mice. NDPK-B(-/-) mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B(-/-) mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1(-/-) mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B(-/-) mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B(-/-) mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases.     

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Isolation of constitutive variants of a subfamily 10 histidine protein kinase (SppK) from Lactobacillus using random mutagenesis

The histidine protein kinase SppK is a peptide pheromone-activated kinase that regulates the production of the bacteriocin sakacin P in Lactobacillus sakei. SppK belongs to subfamily 10 of histidine protein kinases (HPKs), which regulate important processes in Gram-positive bacteria, including virulence, competence and bacteriocin production. To obtain insight into the functional properties of this relatively unknown class of HPKs, we have subjected SppK to random mutagenesis by error-prone PCR, followed by selection for mutants displaying a constitutive phenotype. Most identified mutations were clustered in a predicted coiled coil-like region, which is an important part of the HPK dimer interface and which includes the autophosphorylated histidine. Other mutations were located in the junctions between the dimerization domain and the membrane receptor domain or the catalytic kinase domain. Interestingly, two previously identified constitutive variants of ComD, an SppK homologue involved in competence regulation in Streptococcus pneumoniae, contained single mutations in the same regions.     

Interactions of molybdocene dichloride with histidine- and methionine-containing peptides

The reaction behavior of the antitumor active metallocene dihalide Cp₂MoCl₂ (Cp = η⁵-cyclopentadienyl) towards AcHis, AcHis(1-Me), AcHis(3-Me), His-Gly, AcHis-Gly-His, AcMet, Gly-Met-Gly and cyclo-Met-Met has been studied in solution at 2.5 ? pD ? 7.4 by using ¹H NMR spectroscopy. The histidine-containing substrates were found to bind the Cp₂Mo²⁺ unit through the terminal carboxylate group or through the N1 nitrogen of the imidazole ring, depending on the pD value. At physiological pH, coordination takes place exclusively at the imidazole ring with the percentage of Cp₂Mo²⁺-His adducts in 1:1 mixtures being about 70%. By contrast, the thioether group in the side chain of methionine has a very low affinity for the Cp₂Mo²⁺ group. Monodentate S-coordination could not be detected. For AcMet, binding through the carboxylate group was observed as the only coordination mode, while in the case of Gly-Met-Gly Mo-S interaction occurs in combination with carboxylate coordination leading to a S,O-macrochelate in low yield. Coordination to methionine peptides only takes place at pD ? 6, while at physiological pH interactions with the weak donor sites are not observed due to predominating dimerization of [Cp₂Mo(H₂O)(OH)]⁺ to [Cp₂Mo(μ-OH)₂MoCp₂]²⁺. At c(Cl⁻) = 100 mM competitive Cl⁻ coordination reduces the amount of carboxylate and S,O-coordination significantly, while imidazole coordination is not affected.     

Discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in Hydrogenobacter thermophilus

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent phosphoglycerate mutase (dPGM). However, because they possessed neither mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward l-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from "dPGM-like" proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism.     

A novel histidine kinase gene, ZmHK9, mediate drought tolerance through the regulation of stomatal development in Arabidopsis

Plants have developed complex signaling networks to regulate biochemical and physiological acclimation, environmental signals were perceived and transmitted to cellular machinery to activate adaptive responses. Here, a novel drought responsive histidine kinase gene was identified and designated as ZmHK9. Under normal conditions, ZmHK9 was predominantly expressed in roots, and the roots of ZmHK9-OX transgenic lines are markedly hypersensitive to ABA and ethylene, as compare to wild type. Consistent with its expression induced by PEG and exogenous ABA treatment, promoter sequence of this gene possessed drought and ABA responsive element. Moreover, the transgenic plants were much less affected by drought stress and recovered quickly after rewatering, stomatal complex size and stomatal density in the transgenic plants are significantly smaller and lower than those of the wild-type plants. In addition, ABA induced stomatal closure and the stomatal aperture of ZmHK9-OX lines was smaller than that of wild type. Collectively, it can be concluded that ZmHK9 regulates root elongation, stomatal development and drought tolerance through ABA dependent signaling pathway in Arabidopsis.     

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ～6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.     

Reaction of [Pt(Gly-Gly-N,N',O)I]- with the N-acetylated dipeptide L-methionyl-L-histidine: selective platination of the histidine side chain by intramolecular migration of the platinum(II) complex

The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]⁻ complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by ¹H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]⁻ complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]⁻ was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]⁺ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins.