Tyrosine72 Residue at the Bottom of Rim Domain in LukF Crucial for the Sequential Binding of the Staphylococcal γ-Hemolysin to Human Erythrocytes

Staphylococcal bi-component cytotoxins, leukocidin (Luk), Panton-Valentine leukocidin (PVL), and γ-hemolysin (Hlg) consist of LukF and LukS, LukF-PV and LukS-PV, and LukF and Hlg2, respectively, and Luk and Hlg share LukF. LukF-PV can not substitute for LukF for Hlg, despite 73% identity in amino acid sequence and close similarity in the 3-dimensional structure between them. Here, we demonstrated that the absence of hemolytic activity of LukF-PV in cooperation with Hlg2 is due to the failure of the binding of LukF-PV to human erythrocytes. We identified Y72 residue at the bottom of rim domain in LukF as the crucial residue for its binding, which is a prerequisite to the subsequent binding of Hlg2 to human erythrocytes. The data obtained showed that a mutant of LukF-PV in which T71 residue was replaced by the corresponding residue of LukF, Y72, endowed LukF-PV with the binding capability to human erythrocytes which was accompanied by its hemolytic activity in the presence of Hlg2.     

Identification of the Minimum Segment Essential for the Hγll-Specific Function of Staphylococcal γ-Hemolysin

Staphylococcal γ-hemolysin consists of HγI (or LukF) of 34 kDa and HγII of 32 kDa, which cooperatively lyse human erythrocytes. Our previous data showed that the N-terminal 57-residue segment of HγII is the essential region for the HγII function [H. Nariya and Y. Kamio, Biosci Biotech. Biochem., 59, 1603–1604 (1995)]. To identify the minimum amino acid residues in the 57-residue segment responsible for the specific hemolytic activity, a series of mutant genes were constructed and expressed in Escherichia coli. The mutant proteins were purified and assayed for their hemolytic activity. The results indicate that the 5-residue segment (K²³R²⁴L²⁵A²⁶I²⁷) of HγII is the minimum region essential for the HγII function.     

Substitution of Lysine for Arginine in the N-Terminal 217th Amino Acid Residue of the HγII of Staphylococcall γ-Hemolysin Lowers the Activity of the Toxin

The staphyloccocal toxin γ-hemolysin consists of two protein components, LukF and HyII. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and HγII for leukocidin or γ-hemolysin. HγII of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of HγII [HγII(P83)], in which the 217th arginine residue was replaced by lysine. The HγII(P83) showed about 50% of the hemolytic activity of normal HyII in the presence of LukF.     

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the “stem” region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the “stem” region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.     

An Alternative Conformation of the T-Cell Receptor α Constant Region

αβ T-cell receptors (TcRs) play a central role in cellular immune response. They are members of the Ig superfamily, with extracellular regions of the α and β chains each comprising a V-type domain and a C-type domain. We have determined the ectodomain structure of an αβ TcR, which recognizes the autoantigen myelin basic protein. The 2.0-Å-resolution structure reveals canonical main-chain conformations for the V^α, V^β, and C^β domains, but the C^α domain exhibits a main-chain conformation remarkably different from those previously reported for TcR crystal structures. The global IgC-like fold is maintained, but a piston-like rearrangement between BC and DE β-turns results in β-strand slippage. This substantial conformational change may represent a signaling intermediate. Our structure is the first example for the Ig fold of the increasingly recognized concept of “metamorphic proteins.”     

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an M_r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000–1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg⁷⁰⁹/Arg¹⁰¹⁸/Arg¹⁵⁴⁵) mutated to glutamine (fV^Q3), a mutant fV molecule with region 1000–1008 deleted (fV^ΔB9), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV^ΔB9/Q3). The recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV^Q3 was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K_D(app) values for fV^ΔB9 (0.9 nm), fVa^ΔB9 (0.4 nm), and fV^ΔB9/Q3 (0.7 nm) were similar to the affinity of fVa^WT for fXa (0.3 nm). Two-stage clotting assays revealed that although fV^Q3 was deficient in its clotting activity, fV^ΔB9/Q3 had clotting activity comparable with fVa^WT. The k_cat value of prothrombinase assembled with fV^ΔB9/Q3 was minimally affected, whereas the K_m value of the reaction was increased 57-fold compared with the K_m value obtained with prothrombinase assembled with fVa^WT. These findings strongly suggest that amino acid region 1000–1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.     

Introduction of Standard for the operation of European Biological Resource Centers

(Continued)The equipment and environment in the premises must conform to European legislation meeting quality standards as well as health and safely requirements. The following conditions must be met:Ⅰ. BRC laboratories are clean and well itⅡ. No source of excessive or unusual microbial contamination is introducedⅢ. Adequate bench and storage space is provided, consistent with the type and volume of work,Ⅳ. An appropriate containment facility is available to protect the work and worker from potential release of aerosols containing microorganisms or hazardous chemicals.     

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.     

Scanning Mutagenesis of ��-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the ��9��10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp⁵–Arg⁷ and Asp¹¹–Ile¹⁵, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.Marine snails belonging to the Conus genus produce a variety of neurotoxic peptides in their venom glands that they use for the capture of prey (1–3). Within this repertoire of conopeptides, those that are disulfide-rich are referred to as conotoxins. Conotoxins typically range in size from 12 to 30 amino acids, contain 4 or more Cys residues, and exhibit high potency and selectivity toward a variety of membrane receptors and ion channels (4, 5). The α-conotoxin subfamily members typically range in size from 12 to 19 amino acids, contain 2 disulfide bonds in a Cys^I–Cys^III and Cys^II–Cys^IV connectivity, and have an amidated C terminus, as depicted in Fig. 1. They interact with nicotinic acetylcholine receptors (nAChRs),⁴ of both the muscle and the neuronal type, which have been implicated in a range of neurological disorders varying from Alzheimer disease to addiction (6–8).Open in a separate windowFIGURE 1.α-Conotoxin sequences and structure of Vc1.1. a, the sequences of selected α-conotoxins relevant to this study are shown by one-letter amino acid codes. The asterisk indicates an amidated C terminus, which is a common post-translational modification found in α-conotoxins. The conserved cysteine residues are highlighted in yellow, and the Cys^I–Cys^III and Cys^II–Cys^IV disulfide connectivity is indicated by the connecting lines under the sequence. The number of residues between the cysteines define two backbone “loops,” which are used to classify α-conotoxins into subclasses. For example, RgIA has four residues in loop 1 and three residues in loop 2, making this a 4/3 loop subclass α-conotoxin. b, structural representation of Vc1.1 (PDB 2H8S), with disulfide bonds depicted in yellow. The cysteines, the loops, and the termini are labeled.The nAChRs are ligand-gated ion channels that respond to ACh, nicotine, and other competitive agonists/antagonists. They are composed of five subunits, with differing nAChR subunit composition according to the site of expression. The muscle-type nAChRs are composed of two α subunits, a β and δ subunit, and either an ϵ or a γ subunit (9–12). The neuronal forms exist either as homomeric channels composed of α subunits alone or αβ heteromeric channels. The wide variety of possible subunit combinations has led to unique subtypes with distinct pharmacological properties. This makes α-conotoxins valuable neuropharmacological tools and drug leads, because they have the ability to distinguish between different nAChR subtypes. Effectively, they are small rigid scaffolds that display amino acids on their surface to selectively target their receptors (13).Of particular interest in this study is the α-conotoxin Vc1.1, a synthetic derivative of a naturally occurring peptide from the venom of the marine cone snail, Conus victoriae. It was discovered using PCR screening of cDNA extracted from the snail venom duct (14). Fig. 1 depicts the sequences of selected α-conotoxins, including Vc1.1, which is 16 amino acids in length and displays the classic disulfide bond connectivity observed for α-conotoxins, together with a short helical segment as depicted in Fig. 1b. The conserved Cys framework of α-conotoxins defines two backbone loops, which vary in size and residue composition, and are classified by an n/m nomenclature to define subclasses of α-conotoxins. For example, Vc1.1 is a 4/7 subclass α-conotoxin, because it contains four residues in loop 1 and seven in loop 2. RgIA (15–17) is another conotoxin of interest in this study, because it is also selective for the α9α10 nAChR subtype, and has a 4/3 framework. Vc1.1 contains an amidated C terminus, a post-translational modification common to most α-conotoxins, but it is not present in RgIA. Vc1.1 lacks the post-translationally modified hydroxyproline and γ-carboxyglutamate residues present in the native peptide, vc1a, isolated from the venom duct of C. victoriae (18).Vc1.1 has been under development as a drug lead for neuropathic pain (19). When tested in rat models of neuropathic pain, Vc1.1 induced analgesia when injected intramuscularly near the site of injury (20). Initially, it was thought that α3-containing subtypes of nAChRs may be the target for Vc1.1 (21); however, it was then reported that Vc1.1 has a 100-fold higher affinity at the α9α10 nAChR subtype (22, 23). The α9α10 nAChR mediates synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (24–26). The mRNA of these receptor subtypes is expressed in many different tissue types from the inner ear, dorsal root ganglion (27), skin keratinocytes (28), and lymphocytes (29) to the pituitary (26). The α10 subunit has to be expressed with the α9 subunit to form a functional receptor. In the auditory system, the α9α10 nAChR plays an important role in hair cell development, but its role in other tissues is yet to be characterized (22, 26, 30, 31).Owing to the promising antinociceptive effects of Vc1.1 in animals, its analogs are of interest as leads for the treatment of neuropathic pain (14, 20). To date, studies have predominantly focused on the α9α10 nAChR, but the very recent finding that Vc1.1 also targets the γ-aminobutyric acid, type B receptor (32) has raised interest in the molecular mode of action of Vc1.1 in analgesia. Hence there is a need to define structure-activity relationships of this peptide at several targets, including human and rat forms of the α9α10 nAChR. In particular, we were interested in analogs that maintain potency at the rat α9α10 nAChR but also show significant improvement in potency at human forms of the receptor, while maintaining selectivity over other nAChR subtypes.In this study we determined such structure-activity relationships for Vc1.1 at the α9α10 nAChR by successively mutating each non-Cys residue of Vc1.1 to either an “inert” residue (Ala), a negatively charged residue (Asp), or a positively charged residue (Lys) and observing the impact on the structure and functional activity of Vc1.1. Once the key residues had been identified, a second generation of analogs with new substitutions was synthesized and tested at the rat α9α10 nAChR. The analogs were also analyzed at the human α9/rat α10 (hα9rα10) hybrid clone, because a recent report⁵ suggested differences in the activity of Vc1.1 at the human and rat clones of the α9α10 nAChR. We also examined the effect of pH change on the structure of Vc1.1 using NMR αH chemical shift analysis. The results from this study provide valuable insight into the key residues involved in the interaction of Vc1.1 with the α9α10 nAChR subtype and have the potential to assist in the development of conotoxin analogs as drug leads for the treatment of neuropathic pain (4, 33).     

Human-to-Bovine Jump of Staphylococcus aureus CC8 Is Associated with the Loss of a β-Hemolysin Converting Prophage and the Acquisition of a New Staphylococcal Cassette Chromosome

Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.     

Effect of Zn（Ⅱ） on the Structure and Biological Activity of Natural β-NGF

Only β-NGF, the subunit of the 7S NGF complex, exhibits NGF activity, but the function ofthe zinc ion in native β-NGF has received little attention. Flameless atomic absorption spectroscopy (FAAS)measurements reveal that native β-NGF contains Zn(Ⅱ) with a Zn(Ⅱ)/β-NGF stoichiometry of 1 : 14.6.The presence of Zn(Ⅱ) in the native molecule results in significant changes of the secondary structure andlocal tertiary structure around Trp(s) with respect to those of apo β-NGF, as suggested by spectra offluorescence and circular dichrosim. Stopped-flow studies show that there are at least two steps during theinteraction of Zn(Ⅱ) with the apo form. In comparison with its apo form, the native β-NGF shows a higherability to trigger the proliferation of TF1 cells and mediate the survival of PC 12. Thus it is most likely that thestructural changes caused by the presence of Zn(II) directly lead to the increase in the biological activity of β-NGE All results indicate that Zn(Ⅱ) in native β-NGF plays an important role in the structure and thebiological activity of the protein.     

Regulation of Argonaute Slicer Activity by Guide RNA 3′ End Interactions with the N-terminal Lobe

Structural studies indicate that binding of both the guide RNA (siRNA and miRNA) and the target mRNA trigger substantial conformational changes in the Argonaute proteins. Here we explore the role of the N-terminal lobe (and its PAZ domain) in these conformational changes using biochemical and cell culture-based approaches. In vitro, whereas deletion (or mutation) of the N-terminal lobe of DmAgo1 and DmAgo2 had no effect on binding affinity to guide RNAs, we observed a loss of protection of the 3′ end of the guide RNA and decreased target RNA binding; consistent with this, in cells, loss of function DmAgo1 PAZ variant proteins (PAZ6 and ΔN-PAZ) still bind RNA, although the RNAs are shorter than normal. We also find that deletion of the N-terminal lobe results in constitutive activation of endogenous PIWI domain-based cleavage activity in vitro, providing insights into how cleavage activity may be regulated in vivo in response to different types of pairing interactions with the target mRNAs.     

India’s Biological Diversity Act 2002: An act for the new millenium

Journal of Biosciences -