Α-helical Fold Research Articles

Triintsin, a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity

Since there is a symbiotic and competitive relationship between microorganisms in the same ecological niche, fungal defensins have been found to be important resources for antimicrobial peptides. Here, a fungal defensin, triintsin, was characterized in a clinical isolate of Trichophyton interdigitale from a patient with onychomycosis. The comparison of its genomic and mRNA sequences showed the gene organization and structure of three coding exons separated by two introns. The precursor peptide of triintsin contained 85 amino acid residues, which were composed of three parts including an N-terminal signal domain of 21 residues, a pro-peptide of 47 residues that ended at lysine-arginine and a mature peptide of 38 residues at the C-terminus. The 3D-structure established by homology modeling revealed that triintsin presented a representative typical cysteine-stabilized α-helical and β-sheet fold. The reductive linear peptide of triintsin was obtained by chemical synthesis. After cyclization to form three pairs of disulfide bonds, the oxidative-type peptide displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria but also showed anti-fungal activity. Moreover, triintsin can effectively inhibit the growth of clinical strains. Altogether, the peptide is a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds.

Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form β-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αβ folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αββ fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.

Identification of the Autochaperone Domain in the Type Va Secretion System (T5aSS): Prevalent Feature of Autotransporters with a β-Helical Passenger.

Autotransporters (ATs) belong to a family of modular proteins secreted by the Type V, subtype a, secretion system (T5aSS) and considered as an important source of virulence factors in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria). While exported by the Sec pathway, the ATs are further secreted across the outer membrane via their own C-terminal translocator forming a β-barrel, through which the rest of the protein, namely the passenger, can pass. In several ATs, an autochaperone domain (AC) present at the C-terminal region of the passenger and upstream of the translocator was demonstrated as strictly required for proper secretion and folding. However, considering it was functionally characterised and identified only in a handful of ATs, wariness recently fells on the commonality and conservation of this structural element in the T5aSS. To circumvent the issue of sequence divergence and taking advantage of the resolved three-dimensional structure of some ACs, identification of this domain was performed following structural alignment among all AT passengers experimentally resolved by crystallography before searching in a dataset of 1523 ATs. While demonstrating that the AC is indeed a conserved structure found in numerous ATs, phylogenetic analysis further revealed a distribution into deeply rooted branches, from which emerge 20 main clusters. Sequence analysis revealed that an AC could be identified in the large majority of SAATs (self-associating ATs) but not in any LEATs (lipase/esterase ATs) nor in some PATs (protease autotransporters) and PHATs (phosphatase/hydrolase ATs). Structural analysis indicated that an AC was present in passengers exhibiting single-stranded right-handed parallel β-helix, whatever the type of β-solenoid, but not with α-helical globular fold. From this investigation, the AC of type 1 appears as a prevalent and conserved structural element exclusively associated to β-helical AT passenger and should promote further studies about the protein secretion and folding via the T5aSS, especially toward α-helical AT passengers.

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (NTAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, NTAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the NTAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both NTAIL/XD and NTAIL/hsp70 interactions. Although the same NTAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased NTAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.

Invasive fungi-derived defensins kill drug-resistant bacterial pathogens

Fungi-derived defensins are a class of antimicrobial peptides with therapeutic potential due to their high antibacterial efficacy and low toxicity. Based on the genomic strategy, we have identified 68 fungal defensin-like peptides (fDLPs) in five new genera, including Trichosporon, Apophysomyces, Lichtheimia, Beauveria and Scedosporium and characterized a new synthetic defensin (scedosporisin) from an invasive fungus. It was active against Gram-positive bacteria but not active against negative bacteria. Importantly, it killed several clinical resistant isolates such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci at low molecular concentrations. Scedosporisin showed low hemolysis and cytotoxicity and high serum stability. The killing kinetics of scedosporisin-2 against a clinical isolate of MRSA showed that it killed the bacteria more rapidly than that of vancomycin. Homology modeling analysis show that scedosporisin adopted a typical cysteine stabilized α-helical and β-sheet fold with a local hydrophobic patch. Scedosporisin significantly improved the survival rate of mice in the peritonitis model. This work has greatly expanded the library of fDLPs, and successfully selected leading molecules for antimicrobial drug reserves.

TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition

Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.

Structure-Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis.

The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A.tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A.tridentata FPPase and CPPase are located within a six-helix bundle containing amino acids 53 to 241. The two enzymes were metamorphosed into one another by sequentially replacing the loops and helices of the six-helix bundle from enzyme with those from the other. Chain elongation was the dominant activity during the N-terminal to C-terminal metamorphosis of FPPase to CPPase, with product selectivity gradually switching from FPP to GPP, until replacement of the final α-helix, whereupon cyclopropanation and branching activity competed with chain elongation. During the corresponding metamorphosis of CPPase to FPPase, cyclopropanation and branching activities were lost upon replacement of the first helix in the six-helix bundle. Mutations of active site residues in CPPase to the corresponding amino acids in FPPase enhanced chain-elongation activity, while similar mutations in the active site of FPPase failed to significantly promote formation of significant amounts of irregular monoterpenes. Our results indicate that CPPase, a promiscuous enzyme, is more plastic toward acquiring new activities, whereas FPPase is more resistant. Mutations of residues outside of the α terpene synthase fold are important for acquisition of FPPase activity for synthesis of CPP, LPP, and MPP.

Identification of Key Interactions in the Initial Self-Assembly of Amylin in a Membrane Environment

Islet amyloid polypeptide, also known as amylin, forms aggregates that reduce the amount of insulin-producing cells in patients with type II diabetes mellitus. Much remains unknown about the process of aggregation and cytotoxicity, but it is known that certain cell membrane components can alter the rate of aggregation. Using atomistic molecular dynamics simulations combined with the highly mobile membrane mimetic model incorporating enhanced sampling of lipid diffusion, we investigate interaction of amylin peptides with the membrane components as well as the self-assembly of amylin. Consistent with experimental evidence, we find that an initial membrane-bound α-helical state folds into stable β-sheet structures upon self-assembly. Our results suggest the following mechanism for the initial phase of amylin self-assembly. The peptides move around on the membrane with the positively charged N-terminus interacting with the negatively charged lipid headgroups. When the peptides start to interact, they partly unfold and break some of the contacts with the membrane. The initial interactions between the peptides are dominated by aromatic and hydrophobic interactions. Oligomers are formed showing both intra- and interpeptide β-sheets, initially with interactions mainly in the C-terminal domain of the peptides. Decreasing the pH to 5.5 is known to inhibit amyloid formation. At low pH, His18 is protonated, adding a fourth positive charge at the peptide. With His18 protonated, no oligomerization is observed in the simulations. The additional charge gives a strong midpoint anchoring of the peptides to negatively charged membrane components, and the peptides experience additional interpeptide repulsion, thereby preventing interactions.

Nitrophorins and nitrobindins: structure and function.

Classical all α-helical globins are present in all living organisms and are ordered in three lineages: (i) flavohemoglobins and single domain globins, (ii) protoglobins and globin coupled sensors and (iii) truncated hemoglobins, displaying the 3/3 or the 2/2 all α-helical fold. However, over the last two decades, all β-barrel and mixed α-helical-β-barrel heme-proteins displaying heme-based functional properties (e.g. ligand binding, transport and sensing) closely similar to those of all α-helical globins have been reported. Monomeric nitrophorins (NPs) and α1-microglobulin (α1-m), belonging to the lipocalin superfamily and nitrobindins (Nbs) represent prototypical heme-proteins displaying the all β-barrel and mixed α-helical-β-barrel folds. NPs are confined to the Reduviidae and Cimicidae families of Heteroptera, whereas α1-m and Nbs constitute heme-protein families spanning bacteria to Homo sapiens. The structural organization and the reactivity of the stable ferric solvent-exposed heme-Fe atom suggest that NPs and Nbs are devoted to NO transport, storage and sensing, whereas Hs-α1-m participates in heme metabolism. Here, the structural and functional properties of NPs and Nbs are reviewed in parallel with those of sperm whale myoglobin, which is generally taken as the prototype of monomeric globins.

Deciphering the Molecular Effects of Mutations on ATRX Cause ATRX Syndrome: A Molecular Dynamics Study.

α-thalassemia mental retardation X-linked (ATRX) syndrome is caused by the dysfunction of ATRFfigX protein. The present study explored the structural consequences influenced by two observed mutations V194I and C220R on ADD domain of ATRX protein by applying all atom molecular dynamics (MD) simulation. MD result showed that both the mutants exhibited wide variations in their backbone dynamics, as a result, mutant V210I showed complete distortion on α3 and the mutant C220R displayed a biased disruption on α2-3. The interference in the local folding of α-helices in both the mutants resulted by the loss of hydrogen bonds mediated by the backbone atoms. Principle component analysis (PCA) elucidated that both the mutants endured a diverse conformational dynamics, consequently adopted thermodynamically different conformational state. Besides, binding residues in both the mutants showed more structural disorder, thereby unable to recognize the hallmark modification, K9me3 (tri-methylated lysine at position 9) of histone H3 peptide and it was not conducive for the wild type ADD domain like functionality. Altogether, our findings provide knowledge to understand the structural and functional relationship of disease-associated mutations, V194I and C220R on ADD domain as well as gain further insights into the molecular pathogenesis of ATRX syndrome. J. Cell. Biochem. 118: 3318-3327, 2017. © 2017 Wiley Periodicals, Inc.

Conformational motion correlations in the formation of polypeptide secondary structure in a viscous medium

The Langevin dynamics method and statistical correlation analysis were used to study the α-helical structure folding dynamics of the (Ala)50, (AlaGly)25, and (AlaGly)75 polypeptides depending on the viscosity of the medium. Friction forces that arise when the effective viscosity of the medium is similar to the viscosity of water were found to result in strong correlations between the backbone torsion angles. The polypeptides under study folded mainly to produce α-helical structures. A structure of two contacting α-helices that were approximately equal in length and had a loop between them was observed for a longer chain of 150 residues. A method to visualize the correlation matrix of the dihedral angles of a polypeptide chain was developed for analyzing the effects of the dynamic correlation of conformational degrees of freedom. The analysis of the dynamics of the correlation matrix showed that rotations involving angles of the same type (φ–φ and ψ–ψ) occur predominantly in one direction. Rotations invoving different angles (φ–ψ) occur predominantly in opposite directions, so that the total macromolecule does not rotate. A significant reduction in the effective viscosity of the medium disrupts the correlation and makes the rotations stochastic, thus distorting the formation of the regular (helical) structure. The effects of correlated conformational motions are consequences of viscous friction forces. This conclusion agrees with our previous results that outlined the principle of the minimum rate of energy dissipation and the equipartition of energy dissipation rate between conformational degrees of freedom.

Fusion expression of Occludin extracellular loops and an α-helical bundle: A new research model for tight junction.

Tight junctions (TJs) are the outermost structures of intercellular junctions and are highly specialized membrane domains involved in many important cellular processes. However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. Human occludin (OCLN) is a major component of TJs and an essential co-receptor for hepatitis C virus (HCV) cell entry. To explore expression strategy for recombinant TJ proteins possessing integrated and functional extracellular loops, OCLN was here used as a model molecule, and several prokaryotic fusion constructs were designed by docking OCLN extracellular loops (ECLs) to HIV-1 gp41 NHR and CHR six-helical bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated. The proteins were successfully expressed and purified in E. coli, and the double-loop constructs (D1ECL1S+D2ECL2 as a representative) were found to have more potent HCV neutralizing activity than single-loop constructs at non-cytotoxic concentrations. Circular dichroism studies indicate that D1ECL1S+D2ECL2 adopt stable α-helical folds consistent with design. Thermal denaturation assay indicated that D1ECL1S+D2ECL2 is highly stable at 80°C (melting temperature, Tm, of 89.08 ± 2.0°C) and comparable in stability to the 6HV1 scaffold. Moreover, the time-of-addition experiment revealed that D1ECL1S+D2ECL2 predominantly functioned during the early stages of HCV entry. Taken together, these findings provide a novel strategy for recombinant TJ protein expression in vitro, which may shed light on functional and structural studies for TJs and may provide a new avenue to drug development.

Prediction of Metal Ion Binding Site in Truncated Globin of Myxococcus xanthus DK1622 in Homologous Model

Globins are heme containing proteins with the capability to bind oxygen reversibly. All globins can be classified as M (myoglobin-like), S (sensor) and T (truncated) globins; among them, M and S globins have a typical 3/3 α-helical globin fold and T globins have a 2/2 α-helical fold. Till date, all the myxobacterial genomes available in NCBI data base harbor one or more genes for globins but none of the globins from this group of bacteria have been characterized as yet. In this study, in-silico analysis and homology modeling approach was exploited to explore the physiochemical properties and 3-D structure of trHbN (group I T-globin) of Myxococcus xanthusDK1622, a fruiting myxobacteria. After generating the 3-D model, assessment of the structure and stereo-chemical analysis was carried using different evaluation and validation tools.As homologs of the trHbN exist in dimeric as well as monomeric form, dimerization studies were performed and it was observed that dimeric form of the protein is more stable as compared to the monomer. Further, heme group was appended to the model structure of trHbN of M. xanthus by Patchdock web server and model-heme complex was further refined by Firedock.The 3-D model structure will enhance the understanding for functional analysis of experimentally derived crystal structure.

Peptide Targeted by Human Antibodies Associated with HIV Vaccine-Associated Protection Assumes a Dynamic α-Helical Structure.

The only evidence of vaccine-induced protection from HIV acquisition in humans was obtained in the RV144 HIV vaccine clinical trial. One immune correlate of risk in RV144 was observed to be higher titers of vaccine-induced antibodies (Abs) reacting with a 23-mer non-glycosylated peptide with the same amino acid sequence as a segment in the second variable (V2) loop of the MN strain of HIV. We used NMR to analyze the dynamic 3D structure of this peptide. Distance restraints between spatially proximate inter-residue protons were calculated from NOE cross peak intensities and used to constrain a thorough search of all possible conformations of the peptide. α–helical folding was strongly preferred by part of the peptide. A high-throughput structure prediction of this segment in all circulating HIV strains demonstrated that α–helical conformations are preferred by this segment almost universally across all subtypes. Notably, α–helical conformations of this segment of the V2 loop cluster cross-subtype-conserved amino acids on one face of the helix and the variable amino acid positions on the other in a semblance of an amphipathic α–helix. Accordingly, some Abs that protected against HIV in RV144 may have targeted a specific, conserved α–helical peptide epitope in the V2 loop of HIV’s surface envelope glycoprotein.

Insights into the Three-Dimensional Structure of Amorpha-4,11-diene Synthase and Probing of Plasticity Residues.

Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure-function relationships of all residues in the active site to obtain further insight into the ADS mechanism.

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.

We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [(2)H,(13)C,(15)N]-labeled protein are shown to yield narrow (13)C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

Characteristics of a Recombinant 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Comamonas sp. Expressed in Escherichia coli.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBDO) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the bphC gene from Comamonas sp. SMN4, which encodes 2,3-DBDO with His-tag, was cloned into a plasmid pQE30 in E. coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified active 2,3-DBDO showed a single band around 33kDa, corresponding the molecular mass of 2,3-DBDO subunit. Two fractions around 170 and 100kDa were separated in gel filtration chromatography, but only former one (the fraction of 170kDa) has 2,3-DBDO activity. The 2,3-DBDO was reported as the polymeric protein consisted of eight subunits. However, the fraction corresponding octameric protein of 2,3-DBDO was not found in the gel filtration chromatography. The 2,3-DBDO was exhibited the maximum activity at pH 9.0 and was stable at pH 8.0, relatively. The circular dichroism (CD) data showed that 2,3-DBDO had an α-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. However, this high stable folding structure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The enzyme was thermally stable and active up to 40°C. The conformational data by CD spectra were consistent with the stability of 2,3-DBDO by checking the activity. The binding affinity (Km ) for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7, 24μM, 50mM and 625μM, respectively.

Alpha-synuclein and familial variants affect the chain order and the thermotropic phase behavior of anionic lipid vesicles

Alpha-synuclein (aSN) is a presynaptic protein with a pathological role in Parkinson's disease (PD). The mutants A30P, E46K and A53T are involved in PD early-onset forms. aSN is natively unfolded but can self-assemble to oligomers and fibrils and binds anionic membranes in a helical conformation. We study the influence of wild-type (wt) aSN and familial variants on the chain order and thermotropic phase behavior of anionic dimyristoylphosphatidylglycerol (DMPG) bilayers by using electron spin resonance and calorimetry, respectively. The alpha-helical conformation of the proteins in the membrane-bound state is assessed by circular dichroism thermal scans. wt and mutated aSN upon binding to fluid DMPG vesicles progressively increase chain order. Lipid:protein molar binding stoichiometries correspond to 50 for A30P, 35–36 for aSN and A53T, 30 for E46K. The temperature range over which the variants assume the α-helical fold correlates directly with the density of proteins on vesicle surfaces. All variants preserve the characteristic chain flexibility gradient and impart motional restriction in the lipid chain. This is evident at the first CH2 segments and is markedly reduced at the chain termini, disappearing completely for A30P. The proteins slightly reduce DMPG main transition temperature, revealing preferential affinity for the fluid phase, and broaden the transition, promoting gel-fluid phase coexistence. The overall results are consistent with protein surface association in which the degree of binding correlates with the degree of folding and perturbation of the membrane bilayer. However, the degree of binding of monomer to membrane does not correlate directly with aSN toxicity in vivo.

Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21.

Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function. Due to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire. The structure of UL21C and the observation of its RNA-binding ability are the latest additions to the navigational chart that can guide the exploration of the multiple functions of UL21.