Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.     

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface–bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

The membranes of lipid enveloped viruses fuse with cellular membranes at the beginning of infection to deliver their genetic material into cells. For some viruses, fusion is at the cell surface; for others, it occurs following transfer of receptor-bound viruses into endosomes. Influenza viruses are in the second group and the virus glycoprotein involved in both receptor-binding and low-pH–triggered membrane fusion is hemagglutinin (HA). HA is synthesized as a precursor, HA0, that is proteolytically cleaved during virus replication into the two disulphide-linked components of infectious virus hemagglutinin, HA1 and HA2 (1–4). For 14 of the 16 HA subtypes, cleavage is by trypsin-like enzymes (5, 6) at an arginine residue that immediately precedes the N terminus of HA2 (7). For some HA0s of the two remaining subtypes, H5 and H7, the arginine residue at the site of cleavage is part of a furin-recognition sequence (8), the presence of which generally correlates with virus pathogenicity (9–11).The three-dimensional (3D) structures of HA0 and HA, before and after cleavage, differ only near the site of cleavage (12, 13). Nevertheless, cleavage is essential for fusion activity (14, 15). It generates the HA2 N terminus at a conserved hydrophobic sequence, which is called the fusion peptide because its synthetic peptide analogs have membrane fusion activity. It has been envisioned that cleavage and sequestering of the N terminus of the fusion peptide in a conserved pocket primes HA for its response to low pH and that activation of subsequent changes in conformation involves release of the fusion peptide from its buried location (16, 17).We have previously studied conformational changes in cleaved HA following incubation at fusion pH (18) using cryogenic electron microscopy (cryo-EM) and identified intermediates in the process. To investigate the importance of cleavage to the mechanism of HA-mediated membrane fusion, we now study the response of HA0 to incubation at low pH. We find that the HA0 structure is extensively changed at low pH and that, unlike the changes detected in cleaved HA, the changes in HA0 are reversible on reincubation at neutral pH. We compare the structure of HA0 at low pH with its structure at neutral pH and with one of the low-pH cleaved-HA intermediates (state IV), the “extended intermediate.” This particularly prominent intermediate contains a long central trimeric coiled coil that is assumed to deliver the fusion peptides at its N termini to target cell membranes and to form a bridge between the HA-associated virus membrane and the target. We discuss the possibility that, although not directly involved in membrane fusion, the changes in conformation of HA0 at low pH are indicators of early changes in the conformation of cleaved HA that are required for membrane fusion.     

Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion.

A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.     

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.     

Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches

The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution. Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.     

Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2.

The molecular mechanism of hemolysis and fusion by influenza virus in acidic media was studied. First, the effect of trypsin treatment on the activity of fibroblast-grown influenza virus was studied. The results showed that the split form of viral hemagglutinin, HA1 and HA2, but not the precursor, is responsible for the activity. Second, the interaction of egg-grown influenza virus, which contains the split hemagglutinin, with lipid liposomes was studied by spin labeling and electron microscopy. Phospholipid transfer from the viral envelope to the lipid bilayer membrane occurred within 30 s at pH 4.5-5.4. The transfer is largely independent of the lipid composition and the crystalline vs. liquid/crystalline state of the membrane. Virus-induced lysis of liposomes also took place rapidly in the same pH range. Envelope fusion with liposomes occurred at pH 5.2 but not at pH 7.0. These characteristic interactions were similar to those between influenza virus and erythrocytes reported previously. On the other hand, hemagglutinating virus of Japan did not interact with liposomes at neutral pH. These results suggest that protonation of the NH2-terminal segment of the HA2 form causes interaction of the segment with the lipid core of the target cell membrane, leading to hemolysis and fusion.     

Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins

Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway.     

Single-particle kinetics of influenza virus membrane fusion

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Analysis of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.     

pH-triggered,activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge–dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ∼40 kHz, with a total open-state population of ∼20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.     

Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice

A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NA^Min, PR8-HA^Min, and PR8-(NA+HA)^Min (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin–Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD₅₀ values: PR8, 32 plaque-forming units (PFU); HA^Min, 1.7 × 10³ PFU; NA^Min, 2.4 × 10⁵ PFU; (NA+HA)^Min, ≥3.16 × 10⁶ PFU. Remarkably, (NA+HA)^Min was attenuated >100,000-fold, with NA^Min the major contributor to attenuation. In vaccinated mice (NA+HA)^Min was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD₅₀) of 2.4 PFU. Moreover, at a PD₅₀ of only 147 or 237, (NA+HA)^Min conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development.     

Influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion

Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.     

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution.     

Structure of the human cohesin inhibitor Wapl

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl–Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1–SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.     

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation–deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.Thrombotic disease is seriously harmful to human health and is one of the major causes of death in modern society (1). Despite their long-term and widespread use as anticoagulants, heparin, low–molecular-weight heparin (LMWH), and coumarins still have a major unresolved issue: the risk of serious bleeding during therapy (1–3). It is generally recognized that the risk of bleeding associated with these agents is related to the nonselectivity of their anticoagulant activity. Therefore, selective inhibitors of human factor Xa (FXa) and thrombin (FIIa), such as dabigatran, rivaroxaban, and apixaban, which have predictable pharmacokinetics, have recently been developed; however, these agents have not effectively reduced the risk of bleeding in clinical applications (4–7).Components of the intrinsic coagulation pathway are promising targets for antithrombotic therapy because they are important for thrombosis but are not required for hemostasis (1, 8). The development of new anticoagulant agents that inhibit components of the intrinsic pathway and that have a lower risk of causing bleeding has thus become a research focus (9–11). Factor IXa (FIXa), a serine protease, and factor VIIIa (FVIIIa), a protein cofactor, form a Ca²⁺- and phospholipid surface-dependent complex referred to as the intrinsic tenase complex, which efficiently converts zymogen factor X (FX) to FXa (1, 12, 13). Because the intrinsic tenase is the final and rate-limiting enzyme complex in the intrinsic pathway, the development of inhibitors of this enzyme complex is important for meeting clinical demands (1). However, limited progress has been achieved due to the unavailability of selective inhibitors with well-defined structures.Fucosylated glycosaminoglycan (FG; 1 in Fig. 1), which is a complex acidic polysaccharide isolated from sea cucumber, has recently attracted considerable attention because of its various bioactivities (14). Notably, FG has potent anticoagulant and antithrombotic activities due to its inhibition of the intrinsic tenase (15–17). However, the native polysaccharide has side effects such as factor XII (FXII) activation, platelet aggregation, and serious bleeding (18). Nowadays, depolymerization is considered to be an effective method for reducing these adverse effects (19). For over 30 y since its discovery the detailed structures of native FG and its depolymerized products have not been elucidated, because these polysaccharides are heterogeneous, namely they are mixtures of isomers with different molecular weights, and because limitations exist in the available strategies for analyzing such molecules (17, 20). For example, although it is assumed that a single fucose (Fuc) is linked to the C-3 position of glucuronic acid (GlcA) via an α-glycosidic bond, there is no direct evidence excluding the possibility that Fuc may be linked to the C-4 and C-6 positions of N-acetylgalactosamine (GalNAc) and that the Fuc side chain may exist as a di- or trisaccharide side chain (14, 21–24). Because the polydisperse and structurally ambiguous native FG and its ill-defined depolymerized products are not suitable for the precise evaluation of their structure–activity relationships, in general, the purification of FG-derived fragments is crucial for probing these structure–activity relationships with regard to the inhibition of the intrinsic tenase and for elucidating the detailed structure of FG. Detailed knowledge of the structures of fragments and of the native form of FG is also necessary for developing a clinically effective inhibitor of the intrinsic tenase that has fewer side effects. In this work, we prepared a class of homogeneous oligosaccharides using our newly developed selective depolymerization method. Analysis of these oligosaccharides revealed the precise structure of FG. To our delight, some of these oligosaccharides have potent anticoagulant activity by strongly and selectively inhibiting the intrinsic tenase while avoiding such side effects as FXII activation and platelet aggregation. Furthermore, we found that nonasaccharide is the minimum structural unit responsible for the selective inhibition of the intrinsic tenase, and that nonasaccharide strongly inhibits venous thrombus formation without bleeding consequences.Open in a separate windowFig. 1.Preparation of FG-derived oligosaccharides. The purified FG (i.e., 1) was N-deacetylated with hydrazine hydrate (A) (yield 95%). The N-deacetylated 1 was then cleaved with nitrous acid (B), reduced with NaBH₄ (C), dialyzed, lyophilized to prepare 2 (yield 80%), and then fractionated by GPC (D) to obtain the pure oligosaccharides 3–8 (n = 0–5).     

Structure of human enterovirus 71 in complex with a capsid-binding inhibitor

Human enterovirus 71 is a picornavirus causing hand, foot, and mouth disease that may progress to fatal encephalitis in infants and small children. As of now, no cure is available for enterovirus 71 infections. Small molecule inhibitors binding into a hydrophobic pocket within capsid viral protein 1 were previously shown to effectively limit infectivity of many picornaviruses. Here we report a 3.2-Å-resolution X-ray structure of the enterovirus 71 virion complexed with the capsid-binding inhibitor WIN 51711. The inhibitor replaced the natural pocket factor within the viral protein 1 pocket without inducing any detectable rearrangements in the structure of the capsid. Furthermore, we show that the compound stabilizes enterovirus 71 virions and limits its infectivity, probably through restricting dynamics of the capsid necessary for genome release. Thus, our results provide a structural basis for development of antienterovirus 71 capsid-binding drugs.     

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.     

甲型流感病毒血凝素研究进展

甲型流感病毒是一种人兽共患病的病原体,它可以引起季节性流感和人感染动物源性流感等多种疾病,具有传染性强、变异率高、危害性大等特点,一直以来都是监测及研究的重点对象.血凝素作为病毒表面的重要糖蛋白之一,在病毒的宿主嗜性、生命周期、变异进化,防控工作的监测监查以及相关抗流感药物的研发等方面地位突出.本文从形态结构、功能作用...     

Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

The majority of influenza vaccines are based on whole, inactivated virus, but various alternatives have been developed such as adjuvanted hemagglutinin (HA) protein, membrane-extracted HAs forming multimeric rosette-like particles (1), or nanoparticles with spatially controlled HA display (2). The production of class I fusion proteins by recombinant protein expression is challenging because of their intrinsic instability, low expression levels, and failure to form correctly folded trimers. Uncleaved HA ectodomain (HA0) is monomeric and does not induce significant amounts of neutralizing antibodies. Although several antibodies that recognize the trimer interface have been described, suggesting some degree of reversible “breathing” of the HA trimer apex (3–6), the cleaved HA at the viral surface is predominantly in a closed trimeric conformation (7–9). For all current vaccines and emerging recombinant vaccine vectors or nucleic acid vaccines (10), the delivery of a conformationally correct prefusion HA trimer with improved expression, quality, and stability is crucial (11).Like other class I fusion proteins, influenza A HA transforms from a high-energy, metastable prefusion state to a postfusion conformation, a transition, which in case of HA, is triggered by low pH. To become functional, HA0 needs to be proteolytically cleaved (12, 13) into the head domain (HA1) and the membrane-anchored fusion domain (HA2). HA2 consists of the N-terminal refolding region 1 (RR1, residues HA2 1 to 75) containing a heptad repeat motif, the central helix (residues HA2 76 to 105), and the C-terminal refolding region 2 (RR2, residues HA2 106 to 184) (Fig. 1A; residue numbering in SI Appendix, Fig. S1A). Early in the refolding process, the N-terminal part of HA2 containing the fusion peptide and two β-strands in the membrane-proximal region show disorder (14–17) (Fig. 1B). Another unstable region is located at the bend between helices D and E (Fig. 1C), just below the binding pocket of the stabilizing compound Arbidol (18). Early in the refolding process, after low pH triggered the release of the fusion peptide, this long helical structure is straightened, starting from H106 into the space freed by the fusion peptide (14, 15). H106 marks the beginning of RR2 and the location where the long helix subsequently breaks in the postfusion structure (Fig. 1D).Open in a separate windowFig. 1.Influenza HA protein structure. (A) Schematic representation of HA with indicated fusion peptide (FP), refolding regions 1 and 2 (RR1 and RR2), heptad repeat (HR) motif, central helix (CH), transmembrane (TM) domain, and cytoplasmic domain (CD). (B) Structure of prefusion trimeric WT H3-HK68 [PDB identifier 4FNK (22)] in surface representation (gray). The internal protein structure is outlined in the cartoon with one monomer colored according to A. (C) Bend of the helical structure between helices D and E. (D) Conformation of the postfusion monomer [PDB identifier 1QU1 (50)]. (E) Location of histidine switches 1 and 2 (pHS1 and pHS2). The histidines and charged residues forming the switches are shown in space, filling representation in orange for pH switch 1 (pHS1) and yellow for pH switch 2 (pHS2). Fusion inhibitor Arbidol is plotted in blue based on PDB identifier 5T6N (18). pHS1, pHS2, and Arbidol binding to the other protomers were plotted as outline. (F) Details of the structure of the histidine switches pHS1 and pHS2 in the WT HA. H106-K51-E103 linked by hydrogen bonds in pHS2 (Top) and H26-R153-E150 triad in pHS1 (Bottom). Residues belonging to pHS1 and pHS2 are outlined in the same color as in E.Here, we describe the low stability and quality of wild-type (WT) influenza A HAs and their stabilization by a minimal substitution of three residues within the pH-sensitive switches involved in the refolding of the proteins. We show that the approach becomes applicable to a broader panel of HA subtypes when supplemented with a strain-specific mutation of rare amino acids back to subtype consensus, a process we call “repair,” previously applied to improving expression of HIV Env fusion proteins (19, 20).     

DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature’s highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand–RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5′GAG/3′CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target–ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

Traditional drug discovery entails screening a single or a few targets for binding to a library of small molecules (1). The dominant high-throughput screening paradigm is responsible for generating countless new lead molecules. These screening data sets and the accompanying compound library files guide medicinal chemistry by way of off-target effects gleaned from prior screens and deep physicochemical and pharmacokinetic analyses that all library members undergo, ultimately focusing the library on “drug-like” chemical matter (2, 3). High-throughput screens are limited to the same “drug-like” chemical matter (2, 3), leading to the classification of a target as “druggable” or “undruggable.” However, the screened chemical matter, developed for “druggable” protein and enzyme targets, is not appropriate for all potential target classes.Genetic encoding of targets and small molecules has greatly increased both the scope of library property space and the information content obtained from such screens (4–8). DNA-encoded library (DEL) technology permits the design and synthesis of highly diverse (∼10⁶) collections of small molecules, each bound to a structure-encoding DNA tag (4, 5, 9–12). Affinity selection and sequencing of the bound species’ DNA tags affords potent ligands and rich structure–activity data (13, 14). Complementarily, RNA sequencing technology has become a powerful tool for identifying selective RNA ligands (15–19). For example, two-dimensional combinatorial screening (2DCS) presents a library of RNA three-dimensional ( 3D) folds to a small molecule microarray; bound RNAs are excised from the microarray and sequenced (20, 21). Thousands of compounds can be screened for binding to thousands of RNA targets, but each compound must be individually synthesized, purified, and assayed for RNA library binding.Herein, we integrate 2DCS with solid-phase DEL in a massively parallel screening pipeline to probe affinity landscapes between RNA folds and small molecules. In brief, fluorescence-activated cell sorting (FACS) identified DEL beads that specifically bind to RNA folds. Selective small-molecule RNA ligands were isolated in a FACS analysis of DEL beads (73,728 members) that were incubated with two differentially labeled RNAs: 1) a library displaying the randomized region in a 3 × 3 nucleotide internal loop pattern (3 × 3 ILL; 4,096 members) and 2) a fully base-paired RNA counter screen target. Sequencing and informatic analysis revealed affinity landscapes and candidate target miRNAs for each discovered ligand. A compound with nanomolar affinity for oncogenic primary microRNA-27a (pri-miR-27a) was discovered and shown to inhibit miRNA biogenesis and rescue a migratory phenotype in triple-negative breast cancer (TNBC) cells.     

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.