Critical Role of a Ferritin-Like Protein in the Control of Listeria monocytogenes Cell Envelope Structure and Stability under β-lactam Pressure

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.     

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.     

The Structure of Bacteriophage φCb5 Reveals a Role of the RNA Genome and Metal Ions in Particle Stability and Assembly

The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions.Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.     

Cation–π Interactions in β-Lactamases: The Role in Structural Stability

β-lactam group of antibiotics is the most widely used therapeutic molecules for treating bacterial infections. The main mode of bacterial resistance to β-lactams is by β-lactamases. In the present study, we report our results on the role of cation–π interactions in β-lactamases and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while tyrosine is comparatively higher than phenylalanine and tryptophan in the π group. Our results indicate that cation–π interactions might play an important role in the global conformational stability of β-lactamases.     

Nascent β-Hairpin Formation of a Natively Unfolded Peptide Reveals the Role of Hydrophobic Contacts

Despite the important role of the unfolded states in protein stability, folding, and aggregation, they remain poorly understood due to the lack of residue-specific experimental data. Here, we explore features of the unfolded state of the NTL9 protein by applying all-atom replica-exchange simulations to the two fragment peptides NTL9(1–22) and NTL9(6–17). We found that while NTL9(6–17) is unstructured, NTL9(1–22) transiently folds as various β-hairpins, a fraction of which contain a native β-sheet. Interestingly, despite a large number of charged residues, the formation of backbone hydrogen bonds is concomitant with hydrophobic but not electrostatic contacts. Although the fragment peptides lack a proposed specific contact between Asp⁸ and Lys¹², the individually weak, nonspecific interactions with lysines together stabilize the charged Asp⁸, leading to a pK_a shift of nearly 0.5 units, in agreement with the NMR data. Taken together, our data suggest that the unfolded state of NTL9 likely contains a β-hairpin in segment 1–22 with sequence-distant hydrophobic contacts, thus lending support to a long-standing hypothesis that the unfolded states of proteins exhibit native-like topology with hydrophobic clusters.     

Application of On-Line nanoLC-IT-TOF in the Identification of Serum β-Catenin Complex in Mice Scald Model

Severe burn shock remains an unresolved clinical problem with an urgent need to explore novel therapeutic treatments. Intracellular β-catenin, through interaction with other proteins, has been reported to be able to regulate the size of cutaneous wounds. Higher expression of β-catenin is associated with larger sized wounds. However, the identification of serum β-catenin complex is difficult and has been rarely reported. The exploitation of more binding partners can contribute to uncovering the exact mechanisms behind serum β-catenin mediated biological effects. Here, we describe a method that consists of immunoprecipitation, SDS-PAGE, in-gel digestion, and nanoLC coupled to LCMS-IT-TOF for the investigation of serum β-catenin complex in mice scald model. Among selected gel bands obtained from the protein gels, a total of 31 peptides were identified and sequenced with high statistical significance (p<0.01). Three proteins (alpha-2-marcoglobulin, serine protease inhibitor A3K, and serine protease inhibitor A1A) were identified and validated with high reliability and high reproducibility. It was inferred that these proteins might interact with serum β-catenin, which could affect the wound healing resulting from burn shock. Our study demonstrated that the on-line coupling of nano-LC with a LCMS-IT-TOF mass spectrometer was capable of sensitive and automated characterization of the serum β-catenin complex in mice scald model.     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.     

Structural Basis for the Function of the β-Barrel Assembly-Enhancing Protease BepA

The β-barrel assembly machinery (BAM) complex mediates the assembly of β-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of β-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.     

Critical Role of Cys168 in Noggin Protein＇s Biological Function

Previous studies have indicated that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin‘s biological activities. It probably participates in the formation of an intermolecular disulfide bridge.     

Structural Analysis of Mutations in the Drosophila β2-Tubulin Isoform Reveals Regions in the β-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

We have determined the lesions in a number of mutant alleles of βTub85D, the gene that encodes the testis-specific β2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the β2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all β-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate β-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that β3, a developmentally regulated Drosophila β-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type β2-tubulin. We show here by complementation analysis that β3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the β2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the β2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the β2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate β-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type β-tubulins. We propose that the integrity of this structure in the Drosophila testis β2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.     

A Retroviral Chimeric Capsid Protein Reveals the Role of the N-Terminal β-Hairpin in Mature Core Assembly

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.     

Brucella Cyclic β-1,2-Glucan Plays a Critical Role in the Induction of Splenomegaly in Mice

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic β-1,2-glucan (CβG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CβG mutants of Brucella, cgs (CβG synthase), cgt (CβG transporter) and cgm (CβG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CβG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CβG in promoting splenomegaly in mice. We showed that CβG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CβG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.     

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.     

Identification and Validation of a Linear Protective Neutralizing Epitope in the β-Pore Domain of Alpha Toxin

The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.     

Molecular Basis for the Structural Stability of an Enclosed β-Barrel Loop

We present molecular dynamics simulation studies of the structural stability of an enclosed loop in the β domain of the Escherichia coli O157:H7 autotransporter EspP. Our investigation revealed that, in addition to its excellent resistance to thermal perturbations, EspP loop 5 (L5) also has remarkable mechanical stability against pulling forces along the membrane norm. These findings are consistent with the experimental report that EspP L5 helps to maintain the permeability barrier in the outer membrane. In contrast to the major secondary structure elements of globular proteins such as ubiquitin, whose resistance to thermal and mechanical perturbations depends mainly on backbone hydrogen bonds and hydrophobic interactions, the structural stability of EspP L5 can be attributed mainly to geometric constraints and side-chain interactions dominated by hydrogen bonds. Examination of B-factors from available high-resolution structures of membrane-embedded β barrels indicates that most of the enclosed loops have stable structures. This finding suggests that loops stabilized by geometric constraints and side-chain interactions might be used more generally to restrict β-barrel channels for various functional purposes.     

Identification of MYO18A as a Novel Interacting Partner of the PAK2/βPIX/GIT1 Complex and Its Potential Function in Modulating Epithelial Cell Migration

The p21-activated kinase (PAK) 2 is known to be involved in numerous biological functions, including the regulation of actin reorganization and cell motility. To better understand the mechanisms underlying this regulation, we herein used a proteomic approach to identify PAK2-interacting proteins in human epidermoid carcinoma A431 cells. We found that MYO18A, an emerging member of the myosin superfamily, is a novel PAK2 binding partner. Using a siRNA knockdown strategy and in vitro binding assay, we discovered that MYO18A binds to PAK2 through the βPIX/GIT1 complex. Under normal conditions, MYO18A and PAK2 colocalized in lamellipodia and membrane ruffles. Interestingly, knockdown of MYO18A in cells did not prevent formation of the PAK2/βPIX/GIT1 complex, but rather apparently changed its localization to focal adhesions. Moreover, MYO18A-depleted cells showed dramatic changes in morphology and actin stress fiber and membrane ruffle formation and displayed increases in the number and size of focal adhesions. Migration assays revealed that MYO18A-depleted cells had decreased cell motility, and reexpression of MYO18A restored their migration ability. Collectively, our findings indicate that MYO18A is a novel binding partner of the PAK2/βPIX/GIT1 complex and suggest that MYO18A may play an important role in regulating epithelial cell migration via affecting multiple cell machineries.     

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3–5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl^−/− background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl^−/− retinas, RDS and ROM-1 levels were decreased by ∼60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.