Zn2+ binding to human calbindin D(28k) and the role of histidine residues

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.     

Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.     

Are aromatic carbon donor hydrogen bonds linear in proteins?

Proteins fold and maintain structure through the collective contributions of a large number of weak, noncovalent interactions. The hydrogen bond is one important category of forces that acts on very short distances. As our knowledge of protein structure continues to expand, we are beginning to appreciate the role that weak carbon-donor hydrogen bonds play in structure and function. One property that differentiates hydrogen bonds from other packing forces is propensity for forming a linear donor-hydrogen-acceptor orientation. To ascertain if carbon-donor hydrogen bonds are able to direct acceptor linearity, we surveyed the geometry of interactions specifically involving aromatic sidechain ring carbons in a data set of high resolution protein structures. We found that while donor-acceptor distances for most carbon donor hydrogen bonds were tighter than expected for van der Waals packing, only the carbons of histidine showed a significant bias for linear geometry. By categorizing histidines in the data set into charged and neutral sidechains, we found only the charged subset of histidines participated in linear interactions. B3LYP/6-31G**++ level optimizations of imidazole and indole-water interactions at various fixed angles demonstrates a clear orientation dependence of hydrogen bonding capacity for both charged and neutral sidechains. We suggest that while all aromatic carbons can participate in hydrogen bonding, only charged histidines are able to overcome protein packing forces and enforce linear interactions. The implications for protein modeling and design are discussed.     

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional ¹³C‐edited ¹H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

QS-type bacterial signal molecules of nonpeptide origin

This review classifies and analyzes the literature data on bacterial autoinducers (AI), the signal molecules produced and secreted by bacterial cells and responsible for intercellular communication (quorum sensing, QS). The most important families of nonpeptide AI are discussed, including N-acyl homoserine lactones, derivatives of 2-methyl-2,3,4,5-tetrahydroxy tetrahydrofuran, indole and quinoline derivatives, and adrenalinerelated compounds. The data is provided on the intracellular and membrane receptors specifically binding to AI, as well as on the effector systems that are activated by AI and mediate their regulatory effects. The possible role of some vertebrate hormones (adrenergic agonists, serotonin, etc.) as AI and their effect on bacterial activity are discussed. The data are presented suggesting a high efficiency of AI-based antibacterial preparations, which selectively disrupt the bacterial information network and thus suppress bacterial infections.     

福氏痢疾杆菌组氨酸合成中双功能焦磷酸水解酶和磷酸核糖环化水解酶双功能蛋白的初步研究

福志贺氏菌属是一类革兰氏阴性杆菌,是引起人类细菌性痢疾的主要致病源。以福志贺氏痢疾杆菌2a型301株全基因组为模板、以pET22b-(+)为载体、克隆了一个关键基因：组氨酸合成途径中双功能焦磷酸水解酶和磷酸核糖环化水解酶蛋白（简称：sf2088）。以BL21（DE3）为表达菌株,优化表达条件,获得了可溶表达的蛋白。经过亲和层析和分子筛层析获得目标蛋白。采用分析超速离心和动态光散射实验,对纯化后的蛋白进行稳定聚集状态的条件搜索,结果发现锌离子对稳定其聚合状态很重要。通过正交实验,获得蛋白保持稳定聚集态的条件。这对该酶的进一步研究奠定了基础。     

Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionate substituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (−50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidation states showed significant differences. Additionally, rates of ligand binding and oxidation of both one- and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His–Asn–Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate binding site(s) at the entrance to the heme cavity is discussed.     

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.