From the Cover: Preconfiguration of the antigen-binding site during affinity maturation of a broadly neutralizing influenza virus antibody

Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.     

A broad and potent neutralization epitope in SARS-related coronaviruses

Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, based on the ancestral virus strain (1, 2), confer protective immunity and greatly decrease the incidence of infection, disease severity, and hospitalization from COVID-19. Many SARS-CoV-2 variants have emerged, and the designated variants of concern (VOCs), especially the recent Omicron variants, as well as some variants of interest, are much more resistant to neutralizing antibody (nAb) responses induced by current vaccines (3–8). A vaccine that is highly protective against current SARS-CoV-2 VOCs could potentially provide broader protection against future emerging variants and possibly other sarbecoviruses. However, neutralizing potency and breadth are often somewhat mutually exclusive; the most highly potent nAbs target the ACE2 receptor binding site (RBS) of the spike (S) protein, but most SARS-CoV-2 VOCs have mutations in the RBS that reduce nAb binding and neutralization. Broad binding antibodies, such CR3022, that target other epitopes on the receptor binding domain (RBD) usually have lower neutralization potency (9). Here, we identify a site of vulnerability on the RBD of the SARS-CoV-2 S protein that is targeted by a few diverse antibodies. Importantly, such antibodies, as exemplified by ADG20, compete with receptor binding, exhibit high neutralizing potency and show broad activity to VOCs, including Omicron, to a conserved region that is present also on other SARS-related coronaviruses (CoVs) including SARS-CoV-1, WIV1, and SHC014.     

Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.     

Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.Antibodies in respiratory mucosa are primary mediators of protective immunity against influenza. Notably, preexisting secretory IgA (S-IgA) antibodies can provide immediate immunity by eliminating a pathogen before the virus passes the mucosal barrier (1–3). Parenteral vaccination induces serum IgG but not S-IgA, so vaccine efficacy is limited. In contrast, intranasal administration of an inactivated influenza vaccine elicits both S-IgA and IgG responses, thus improving the protective efficacy of current vaccination procedures (4–8).IgA in human serum exists predominantly in the form of monomers, whereas the majority of IgA in external secretions is present in the form of polymers. These polymeric IgA forms are associated with the extracellular portion of the polymeric Ig receptor, generating a complex (receptor + polymeric IgA) called S-IgA (9). S-IgA primarily corresponds to dimeric IgA, although low levels of some larger polymeric forms, particularly tetramers, are also present (9–15). Polymeric S-IgA has been shown (both in vitro and in experimental animal models) to be more effective than monomeric IgA or IgG for the neutralization of influenza viruses (16–19). However, little is known of the quaternary structures and neutralizing potencies in viral infection of the various forms of polymeric S-IgA in the human nasal mucosa. In this study, the quaternary structures and neutralizing potencies of nasal antibodies against influenza virus were examined using nasal wash samples from healthy adults who had received intranasally administered inactivated influenza vaccines. These nasal wash samples, containing variously sized Igs, were separated by gel filtration chromatography (GFC) and assessed for neutralization activity against influenza virus. The quaternary structures of the nasal IgA induced by intranasally administered inactivated influenza vaccines then were determined using biochemical techniques and high-speed atomic force microscopy (AFM). We found that human nasal IgA comprised at least five quaternary structures, including monomer, dimer, trimer, and tetramer structures, as well as a polymeric form larger than the tetramer structure. Among these forms, the polymeric structure demonstrated higher neutralizing potency against seasonal influenza viruses (H3N2) and highly pathogenic avian influenza virus (H5N1) compared with the dimeric form, suggesting that large polymeric S-IgA antibodies play crucial roles in protective immunity against influenza virus infection of the human upper respiratory tract.     

Structural basis for suppression of a host antiviral response by influenza A virus

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-β mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3′ end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-β pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.     

Herpesvirus gB: A Finely Tuned Fusion Machine

Enveloped viruses employ a class of proteins known as fusogens to orchestrate the merger of their surrounding envelope and a target cell membrane. Most fusogens accomplish this task alone, by binding cellular receptors and subsequently catalyzing the membrane fusion process. Surprisingly, in herpesviruses, these functions are distributed among multiple proteins: the conserved fusogen gB, the conserved gH/gL heterodimer of poorly defined function, and various non-conserved receptor-binding proteins. We summarize what is currently known about gB from two closely related herpesviruses, HSV-1 and HSV-2, with emphasis on the structure of the largely uncharted membrane interacting regions of this fusogen. We propose that the unusual mechanism of herpesvirus fusion could be linked to the unique architecture of gB.     

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.     

Crystal structure of a nematode-infecting virus

Orsay, the first virus discovered to naturally infect Caenorhabditis elegans or any nematode, has a bipartite, positive-sense RNA genome. Sequence analyses show that Orsay is related to nodaviruses, but molecular characterizations of Orsay reveal several unique features, such as the expression of a capsid–δ fusion protein and the use of an ATG-independent mechanism for translation initiation. Here we report the crystal structure of an Orsay virus-like particle assembled from recombinant capsid protein (CP). Orsay capsid has a T = 3 icosahedral symmetry with 60 trimeric surface spikes. Each CP can be divided into three regions: an N-terminal arm that forms an extended protein interaction network at the capsid interior, an S domain with a jelly-roll, β-barrel fold forming the continuous capsid, and a P domain that forms surface spike projections. The structure of the Orsay S domain is best aligned to T = 3 plant RNA viruses but exhibits substantial differences compared with the insect-infecting alphanodaviruses, which also lack the P domain in their CPs. The Orsay P domain is remotely related to the P1 domain in calicivirus and hepatitis E virus, suggesting a possible evolutionary relationship. Removing the N-terminal arm produced a slightly expanded capsid with fewer nucleic acids packaged, suggesting that the arm is important for capsid stability and genome packaging. Because C. elegans-Orsay serves as a highly tractable model for studying viral pathogenesis, our results should provide a valuable structural framework for further studies of Orsay replication and infection.In the past four decades, the nematode Caenorhabditis elegans has become an important model organism for studying various biological processes such as development, metabolism, aging, the cell cycle, and gene regulation (1). Many key biomedical discoveries about common diseases, including Alzheimer’s disease, diabetes type 2, and even depression, were first made in C. elegans (2). It has been estimated that around 70% of human genes have homologs in C. elegans, including the majority of human disease genes and pathways (3).It was not until very recently that Orsay, the first virus that naturally infects C. elegans in the wild, was discovered (4). In addition, two other nematode viruses, Santeuil and Le Blanc, both of which infect C. briggsae, have also been identified (4, 5). Infections by these newly identified viruses cause abnormal intestinal morphologies without obvious effect on longevity or brood size. Orsay viral particles have been observed intracellularly in both intestinal cells and the somatic gonad (4). Sequence analysis indicated that all three nematode viruses are related to viruses in the family Nodaviridae (4, 5). Nodaviruses are nonenveloped RNA viruses that can be further classified as alphanodaviruses and betanodaviruses, which are known to infect insects and fish, respectively (6).Orsay virus has a bipartite, positive-sense RNA genome ∼6.3 kb in size (4). The RNA1 segment of the Orsay virus encodes a predicted RNA-dependent RNA polymerase (RdRP) of 982 amino acids. RNA2 has two ORFs: one coding for a putative capsid protein (CP) and the other for a nonstructural protein δ of unknown function. The protein translation of the Orsay CP uses an AUG-independent, noncanonical initiation mechanism that is likely shared among all three nematode viruses (7). In addition to the viral CP, a CP–δ fusion protein, which is likely generated by ribosomal frameshifting, has been detected in infected cells as well as purified viruses (7). The Orsay CP and RdRP share up to 26–30% amino acid identity with those from betanodaviruses in the Nodaviridae, but the δ protein does not have any sequence homologs in GenBank.The combination of a simple multicellular host organism and a small naturally infecting virus makes C. elegans-Orsay a highly tractable model for studying host–virus interactions (8). To characterize the molecular properties of the newly discovered Orsay virus, we report the crystal structure of an Orsay virus-like particle (VLP), which shows a T = 3 icosahedral symmetry with a total of 60 trimeric spikes. Each CP molecule can be divided into three linear regions, namely the N-terminal arm, the S domain forming the continuous capsid shell, and the P domain, which forms trimeric surface protrusions. The jelly-roll β-barrel in the S domain closely resembles those from small plant RNA viruses. The P domain consists of a distorted β-barrel that is only remotely related to the P1 domain from a norovirus CP (9). The N-terminal arm of the Orsay CP forms an extensive network inside the capsid through a β-annulus structure similar to tomato bushy stunt virus (10). An N-terminally truncated mutant, CP_42–391, produces a slightly expanded capsid with less RNA packaged inside, suggesting important roles for the N-terminal arm in capsid stabilization and genome packaging. Our results also demonstrate that the Orsay capsid is structurally distinct from alphanodaviruses. Together with primary sequence-based phylogeny, these results suggest that the nematode-infecting viruses and betanodaviruses have a common evolutionary history.     

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

Bacteria of the Bacillus cereus family form highly resistant spores, which in the case of the pathogen B. anthracis act as the agents of infection. The outermost layer, the exosporium, enveloping spores of the B. cereus family as well as a number of Clostridia, plays roles in spore adhesion, dissemination, targeting, and germination control. We have analyzed two naturally crystalline layers associated with the exosporium, one representing the "basal" layer to which the outermost spore layer ("hairy nap") is attached, and the other likely representing a subsurface ("parasporal") layer. We have used electron cryomicroscopy at a resolution of 0.8-0.6 nm and circular dichroism spectroscopic measurements to reveal a highly α-helical structure for both layers. The helices are assembled into 2D arrays of "cups" or "crowns." High-resolution atomic force microscopy of the outermost layer showed that the open ends of these cups face the external environment and the highly immunogenic collagen-like fibrils of the hairy nap (BclA) are attached to this surface. Based on our findings, we present a molecular model for the spore surface and propose how this surface can act as a semipermeable barrier and a matrix for binding of molecules involved in defense, germination control, and other interactions of the spore with the environment.     

Structure of a trimeric nucleoporin complex reveals alternate oligomerization states

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13·Nup145C·Nup84 complex, the centerpiece of the heptamer, at 3.2-Å resolution. Nup84 forms a U-shaped α-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C·Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.     

Structural organization of a filamentous influenza A virus

Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.     

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO₂ and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles.     

High-resolution crystal structure reveals molecular details of target recognition by bacitracin

Bacitracin is a metalloantibiotic agent that is widely used as a medicine and feed additive. It interferes with bacterial cell-wall biosynthesis by binding undecaprenyl-pyrophosphate, a lipid carrier that serves as a critical intermediate in cell wall production. Despite bacitracin’s broad use, the molecular details of its target recognition have not been elucidated. Here we report a crystal structure for the ternary complex of bacitracin A, zinc, and a geranyl-pyrophosphate ligand at a resolution of 1.1 Å. The antibiotic forms a compact structure that completely envelopes the ligand’s pyrophosphate group, together with flanking zinc and sodium ions. The complex adopts a highly amphipathic conformation that offers clues to antibiotic function in the context of bacterial membranes. Bacitracin’s efficient sequestration of its target represents a previously unseen mode for the recognition of lipid pyrophosphates, and suggests new directions for the design of next-generation antimicrobial agents.     

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.     

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-Å resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85β, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85β:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85β and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic α-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic α-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85β inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85β (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.     

Structure of BipA in GTP form bound to the ratcheted ribosome

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.Bacterial protein synthesis involves four main translational GTPase (trGTPase) factors: initiation factor 2 (IF2), elongation factors Tu and G (EF-Tu and EF-G), and release factor 3 (RF3). These factors catalyze major steps in translation initiation, elongation (both decoding and mRNA–tRNA complex translocation), and termination, in a GTP-dependent manner. Several additional GTPase factors, including EF4 (formerly known as LepA), BipA, and RelA, have been revealed to be associated with ribosomes under stress conditions (1).Both EF4 and BipA are paralogs of EF-G (1–5). Although EF4 is highly conserved in bacteria (4), deletion of ef4 gene causes no evident phenotype in Escherichia coli under optimal growth conditions (6). However, EF4 was shown to notably improve protein synthesis under stress conditions (7). Qin et al. (2) reported a unique function of EF4 promoting the back translocation of the elongation complex by one codon, hence presumably providing a second chance for EF-G to carry out a correct translocation.BipA (BPI-inducible protein A) gene is highly conserved among bacterial and chloroplast genomes (4) and has been implicated in regulating a variety of cellular processes including bacterial virulence, symbiosis, various stress responses, resistance to host defenses, swarming motility, biofilm, and capsule formation (8–10). As is the case with EF4, BipA is not required under optimal growth conditions but becomes an essential factor for bacterial survival at low temperature, nutrient depletion, and various other stress conditions (1, 9). The diverse nature of these processes underscores the global regulatory properties of BipA. Similarity to classical trGTPases and EF4 led to the speculation that BipA affects translation through directly interacting with the ribosome. For example, wild-type (fully modified) ribosomes seem to depend on BipA for translation of specific mRNAs (11). Furthermore, as with EF4, overexpression of BipA inhibits transfer-messenger mRNA (tmRNA)-dependent peptide tagging activity of nonstop messages on ribosome (6). Thus, BipA likely functions as an elongation factor as well. Consistent with this notion, BipA is able to bind to 70S ribosome in a GTP-dependent manner and its GTPase activity is enhanced in the presence of ribosomes, a characteristic feature of classical trGTPase factors (5, 12). Salmonella enterica BipA has been shown to interact with either 70S ribosomes or 30S subunits depending on the relative abundance of GTP and of the stress alarmone guanosine-3′,5′-bisdiphosphate (ppGpp), respectively (12). In addition, a recent study links BipA to ribosome biogenesis because bipA gene deletion results in perturbed 50S subunit processing and assembly, particularly at low temperatures (13). Although the evidence for BipA involvement in ribosome biosynthesis and/or functioning in translation is mounting, its exact role remains elusive.As a member of the ribosome-dependent trGTPase family, BipA is proposed to share structural similarity with EF4 and EF-G (4, 5). Indeed, all three consist of five domains, of which the N-terminal G domain (nucleotide-binding domain), the β-barrel domain (domain II), and the two α/β-domains (domains III and IV) are topologically equivalent (5) (Fig. 1). EF-G has G′ domain inserted into its G domain and a unique domain IV, whereas unique C-terminal domains (CTDs) are present in BipA and EF4 (Fig. 1). Despite the similarity, the three proteins have distinct functions probably attributed to their varied domain arrangements and ribosome-binding modes.Open in a separate windowFig. 1.Comparison of domain arrangement and overall structure of EF-G, EF4, and BipA. (A) Structures of isolated EF-G and EF4 are obtained from Protein Data Bank (PDB ID codes: 2BM0 and 3CB4, respectively). Structure of BipA apo form is presented. Domain I (green), also known as the G domain, is the nucleotide-binding region. G′ domain insertion (dark blue) is a characteristic feature of the EF-G protein. Domain II (violet) contains the translation factor signature β-barrel motif. Domains III (yellow) and V (sky blue) contain α/β-motifs. EF-G has a unique domain IV (brown), whereas EF4 and BipA have unique C-terminal domains (warm pink and red, respectively). The same color scheme is used throughout this work. (B) Schematic diagram depicting the domain arrangement of EF-G, EF4, and BipA.Extensive structural studies (14–22) of EF-G bound to ribosome have generated a wealth of atomic or near-atomic resolution information on how EF-G, in particular the positioning of its domain IV in ribosome decoding center, facilitates translocation. Mutagenesis study of EF-G revealed that the highly conserved loops I and II of domain IV disrupt the interactions between the decoding center and the codon–anticodon duplex that act as the barrier for mRNA–tRNA complex translocation (23). Structural studies have also shed light on the molecular basis of how EF4 reverses EF-G catalyzed translocation through its CTD reaching into the PTC and interacting with the acceptor stem of the peptidyl-tRNA in the P site (24, 25). In contrast, structures of neither the isolated BipA nor BipA bound to ribosome, which could illuminate the molecular basis of BipA functioning in protein translation, have been characterized yet. Hence, we aimed to structurally characterize the various biologically relevant states of BipA on and off the ribosome, toward a better understanding of the detailed function of BipA. Note that during the revision process, a paper was published reporting the structure of isolated BipA (26).     

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer’s disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ∼18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.     

Feline immunodeficiency virus (FIV) neutralization: a review

One of the major obstacles that must be overcome in the design of effective lentiviral vaccines is the ability of lentiviruses to evolve in order to escape from neutralizing antibodies. The primary target for neutralizing antibodies is the highly variable viral envelope glycoprotein (Env), a glycoprotein that is essential for viral entry and comprises both variable and conserved regions. As a result of the complex trimeric nature of Env, there is steric hindrance of conserved epitopes required for receptor binding so that these are not accessible to antibodies. Instead, the humoral response is targeted towards decoy immunodominant epitopes on variable domains such as the third hypervariable loop (V3) of Env. For feline immunodeficiency virus (FIV), as well as the related human immunodeficiency virus-1 (HIV-1), little is known about the factors that lead to the development of broadly neutralizing antibodies. In cats infected with FIV and patients infected with HIV-1, only rarely are plasma samples found that contain antibodies capable of neutralizing isolates from other clades. In this review we examine the neutralizing response to FIV, comparing and contrasting with the response to HIV. We ask whether broadly neutralizing antibodies are induced by FIV infection and discuss the comparative value of studies of neutralizing antibodies in FIV infection for the development of more effective vaccine strategies against lentiviral infections in general, including HIV-1.     

Unique carbohydrate�Ccarbohydrate interactions are required for high affinity binding between Fc��RIII and antibodies lacking core fucose

Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen–antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate–carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.