Identification of a novel protein binding to hepatitis C virus core protein

Background:  Hepatitis C virus (HCV) core protein is a multi-functional viral protein that interacts with several target proteins of both viral and cellular origin.
Aim and Methods:  To gain insight into the mechanism of action of HCV core protein, we used a yeast two-hybrid system to identify the core protein-interacting cellular targets.
Results:  A cDNA clone encoding an aspartoacylase was obtained, termed aspartoacylase 3 (ACY3). Interaction between ACY3 and HCV core protein was verified using a co-immunoprecipitation assay in vitro , and a mammalian two-hybrid system in vivo . Fluorescence microscopy showed green fluorescence protein-fused ACY3 localized in the cytoplasm.
Conclusion:  Our data suggest that ACY3 is an HCV core binding protein, which may play a role in the development of HCV-associated diseases.     

乙型肝炎病毒前S1蛋白在血清学诊断乙肝病毒复制中的研究

目的：探讨前S1蛋白（Pre—S1）在血清学诊断乙肝病毒复制中的意义。方法：选择慢性乙型肝炎患者血清250例。分别测定每份患者血清中HBV标志物、前S1蛋白和HBV DNA。结果：HBeAg（＋）组中前S1蛋白阳性率97．2％（70／72）,HBV DNA阳性率100％（72／72）。HBeAg（-）组中前S1蛋白阳性率60．1％（107／178）,HBV DNA阳性率63．5％（113／178）。前S1蛋白和HBV DNA的总符合率为89．6%（224／250）,2者差异无统计学意义（P〉0．05）。HBeAg和HBV DNA的总符合率为54．8％（137／250）,2者差异有统计学意义（P〈0．01）。HBsAg（-）中检出前S1蛋白阳性2例。结论：前S1蛋白比HBeAg更敏感地反映HBV复制情况,且可弥补由于HBsAg基因编码区突变造成的漏诊。     

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.Herpesviruses encompass a large family of infectious agents, including important veterinary and human pathogens (1). Among the latter is human cytomegalovirus (HCMV), which can cause serious disease, particularly in immunocompromised individuals and newborns (2). Despite the importance of HCMV in these medically vulnerable populations, currently available treatment options suffer from issues with toxicities, drug resistance, and/or pharmacokinetics (2, 3), motivating the identification of new drug targets.All herpesviruses of mammals, birds, and reptiles undergo a remarkable process known as nuclear egress as part of the viral lifecycle. It is generally accepted that, after assembly in the nucleus, the viral nucleocapsid undergoes envelopment to cross the inner nuclear membrane (INM) followed by deenvelopment to cross the outer nuclear membrane, resulting in release into the cytoplasm for continuation of the virion maturation process (4). Nuclear egress is orchestrated by a highly conserved, heterodimeric nuclear egress complex (NEC), which recruits one or more protein kinases to disrupt the nuclear lamina, permitting access of nucleocapsids to the INM, where the NEC induces budding of the nucleocapsid into the perinuclear space (5–13). In HCMV, the NEC is comprised of UL50, which is an INM protein, and UL53, which is a nucleoplasmic protein that is brought to the INM by its interaction with UL50. These two proteins and their murine CMV (MCMV) homologues, M50 and M53, are essential for replication and nuclear egress (8, 14–17) of their respective viruses. Although a process similar to herpesvirus nuclear egress was recently described for movement of ribonucleoprotein particles during Drosophila myogenesis (18), no host cell homolog of the NEC that would serve as mediator of this mechanism has yet been identified. Furthermore, no structural information currently exists for any NEC subunit across the Herpesviridae family.To gain a better molecular understanding of herpesvirus nuclear egress, we used NMR methods to solve the structure of the conserved half of MCMV M50 and map residues on the surface of M50 that are involved in interactions with the other NEC subunit. We then tested the importance of several of these residues for heterodimerization of both the MCMV and HCMV NECs by looking at the effect of single-alanine mutations in both M50 and UL50 on binding affinity and replication of HCMV by looking at the effect of mutations in the context of NEC localization, nuclear lamina disruption, and virus production. Our results identified a subunit interaction interface with features that suggest that it could be an attractive antiviral drug target.     

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.     

A small-molecule mimic of a peptide docking motif inhibits the protein kinase PDK1

There is great interest in developing selective protein kinase inhibitors by targeting allosteric sites, but these sites often involve protein–protein or protein–peptide interfaces that are very challenging to target with small molecules. Here we present a systematic approach to targeting a functionally conserved allosteric site on the protein kinase PDK1 called the PDK1-interacting fragment (PIF)tide-binding site, or PIF pocket. More than two dozen prosurvival and progrowth kinases dock a conserved peptide tail into this binding site, which recruits them to PDK1 to become activated. Using a site-directed chemical screen, we identified and chemically optimized ligand-efficient, selective, and cell-penetrant small molecules (molecular weight ∼380 Da) that compete with the peptide docking motif for binding to PDK1. We solved the first high-resolution structure of a peptide docking motif (PIFtide) bound to PDK1 and mapped binding energy hot spots using mutational analysis. We then solved structures of PDK1 bound to the allosteric small molecules, which revealed a binding mode that remarkably mimics three of five hot-spot residues in PIFtide. These allosteric small molecules are substrate-selective PDK1 inhibitors when used as single agents, but when combined with an ATP-competitive inhibitor, they completely suppress the activation of the downstream kinases. This work provides a promising new scaffold for the development of high-affinity PIF pocket ligands, which may be used to enhance the anticancer activity of existing PDK1 inhibitors. Moreover, our results provide further impetus for exploring the helix αC patches of other protein kinases as potential therapeutic targets even though they involve protein–protein interfaces.Protein kinases are a rich source of targets for the development of chemical probes and therapeutics; however, the remarkable similarity of their ATP-binding pockets presents a formidable challenge for the development of selective ATP-competitive inhibitors. Previous efforts to address these limitations have focused on targeting allosteric sites in kinases. Exquisitely selective allosteric inhibitors of the protein kinases AKT, MEK, and ABL are now in clinical trials for cancer, and various other allosteric kinase inhibitors and activators are in preclinical development (1). Despite these recent successes, finding allosteric modulators remains challenging, because most allosteric opportunities are the sites of protein–protein or protein–peptide interactions, which are very difficult to mimic with small molecules. Moreover, traditional chemical screening approaches most often identify ligands for the more druggable ATP-binding pocket.The helix αC patch is an ancient allosteric site present on various serine/threonine and tyrosine kinases (2). The binding of effector proteins to the helix αC patch activates some kinases and inhibits others. The helix αC patch is seen most frequently in the AGC family of serine/threonine kinases, where this site is known specifically as the PDK1-interacting fragment (PIF) pocket. A hydrophobic motif (HM) found in the C-terminal tail of most AGC kinases must bind in cis to the PIF pocket for the kinase to be fully active; however, the AGC kinase PDK1 lacks its own HM, and instead uses its PIF pocket as a docking site to recruit, phosphorylate, and thereby activate 23 other AGC kinases, including AKT, S6K, SGK, RSK, and PKC isoforms (3). The known role of PDK1 as a master regulator of these progrowth and prosurvival kinases has motivated the development of numerous PDK1 inhibitors as potential anticancer agents (4). One strategy for inhibiting PDK1 has been to identify compounds that bind to its PIF pocket and disrupt the recruitment of substrates.Early biochemical studies revealed that PIFtide, a synthetic peptide derived from the HM of the protein kinase PRK2, stimulates PDK1 activity toward a short peptide substrate (5) but disrupts recruitment and phosphorylation of the full-length substrates S6K and SGK (6). Small-molecule mimics of PIFtide have been discovered through pharmacophore modeling (7) and fragment-based approaches (8–10), and some optimized analogs have been characterized structurally (10–13); however, these compounds have limited membrane permeability, which diminishes their utility as chemical probes. Moreover, the lack of a structure of PIFtide bound to PDK1 has impeded the structure-based design of improved analogs that mimic the native allosteric interaction.We have explored various site-directed methods for targeting the PIF pocket of PDK1. Previously, we used a technique known as disulfide trapping (or tethering) to identify small-molecule fragments (molecular weight <250 Da) that inhibit or activate PDK1 by covalently labeling a cysteine residue that was engineered into the PIF pocket (10). Here we sought to discover noncovalent small molecules that could be used as chemical probes of PIF pocket function in cells. We developed a PIFtide competitive binding assay to perform a site-directed screen of ∼154,000 compounds for new PIF pocket ligands. We discovered a series of diaryl sulfonamides (molecular weight ∼380 Da) that were chemically optimized and then characterized biochemically, structurally, and in cells. We also solved the first structure of PIFtide bound to PDK1, which reveals how small molecules mimic this peptide effector and provides insights into the structure-based design of improved PIF pocket ligands. Remarkably, we found that PIF pocket ligands sensitize PDK1 to inhibition by an ATP-competitive inhibitor, enabling more complete suppression of downstream signaling in cells.     

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.     

Crystal structure of human prion protein bound to a therapeutic antibody

Prion infection is characterized by the conversion of host cellular prion protein (PrP^C) into disease-related conformers (PrP^Sc) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP^C rather than PrP^Sc. We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP^C and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP^C conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular β-sheet. The importance of this residue in mediating protein–protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.     

Discovery of New Small Molecule Hits as Hepatitis B Virus Capsid Assembly Modulators: Structure and Pharmacophore-Based Approaches

Hepatitis B virus (HBV) capsid assembly modulators (CpAMs) have shown promise as potent anti-HBV agents in both preclinical and clinical studies. Herein, we report our efforts in identifying novel CpAM hits via a structure-based virtual screening against a small molecule protein-protein interaction (PPI) library, and pharmacophore-guided compound design and synthesis. Curated compounds were first assessed in a thermal shift assay (TSA), and the TSA hits were further evaluated in an antiviral assay. These efforts led to the discovery of two structurally distinct scaffolds, ZW-1841 and ZW-1847, as novel HBV CpAM hits, both inhibiting HBV in single-digit µM concentrations without cytotoxicity at 100 µM. In ADME assays, both hits displayed extraordinary plasma and microsomal stability. Molecular modeling suggests that these hits bind to the Cp dimer interfaces in a mode well aligned with known CpAMs.     

乙肝病毒外膜大蛋白检测对于判定HBV DNA复制的意义

目的:通过检测患者血清乙肝病毒外膜大蛋白(HBV-LP)、HBV DNA以及乙肝病毒标志物(HBV M),探讨HBV-LP对于反映体内乙肝病毒复制的意义．方法:采用荧光定量PCR方法对254份HBV 感染血清的HBV DNA进行检测,并采用酶联免疫吸附试验(ELISA)的方法对HBV-LP、 Pre-S1蛋白及HBV M进行检测．结果:大蛋白的检出结果与HBV DNA的检出结果无明显差异．不同HBV M模式的HBV DNA与HBV-LP的检出结果均无显著性差异． HBV DNA拷贝数的对数值与HBV-LP表达具有相关关系(r=0．945,P<0．001),HBV DNA拷贝数的对数值变化的89%可以用HBV-LP A值为解释变量的线性回归模型来解释．结论:HBV-LP能够反应HBV的复制情况,血清中HBV-LP的含量与HBV DNA的拷贝数具有较好的相关性．     

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T₃₇/T₄₆, followed by T₇₀ and S₆₅. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, ⁵⁴YXXXXLΦ⁶⁰ (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m⁷GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1_50–84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S₆₅ and T₇₀). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.Translation control of gene expression allows cells to respond quickly to external cues. In eukaryotic cells, this regulation occurs mainly at the translation initiation step (reviewed in ref. 1). Cellular eukaryotic mRNAs have a cap structure at their 5′ terminus, which is a modified nucleotide (7-methylguanosine triphosphate, m⁷GpppN, where N is any nucleotide) (2). The translational preinitiation complex assembles at the m⁷GpppN cap via the translation initiation complex 4F (eIF4F) (3), which comprises a cap-binding protein, eIF4E, a DEAD-Box RNA helicase, eIF4A, and a large scaffold protein, eIF4G. The scaffold protein eIF4G interacts with eIF4E through a consensus motif, YXXXXLΦ, where X is any amino acid and Φ is a hydrophobic residue. This motif is also shared by eIF4E binding proteins (4E-BPs). The interaction between eIF4E and 4E-BP is phosphorylation-dependent (4–8). When hypophosphorylated, 4E-BP binds tightly to eIF4E. Hyperphosphorylation of 4E-BP, however, decreases its affinity for eIF4E, enabling eIF4G to interact with eIF4E.Altered regulation of translation initiation has been linked to prion formation (9) and to several human diseases, including autism (10) and cancer (11). eIF4E is overexpressed in a variety of tumor cells (12, 13). This overexpression has been implicated in oncogenic transformation (14, 15), a process that 4E-BPs can effectively revert (14–16). Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and its analogs, exert antitumor activity by suppressing 4E-BP1’s phosphorylation, thus enabling its interaction with eIF4E (17). The ability of 4E-BPs to compete with eIF4G for eIF4E binding is explained by the shared YXXXXLΦ binding motif (18, 19). Crystal structures of mouse eIF4E complexed with either 4E-BP1_51–64, eIF4G-I_569–580, or eIF4G-II_621–637, all short fragments containing the consensus-binding motif, are virtually identical (19–22). NMR spectroscopy titration experiments (23) and small angle X-ray scattering of full-length 4E-BP1 bound to eIF4E (24) suggested that 4E-BP1 has a larger binding interface on eIF4E than eIF4G. In agreement with this observation, mutagenesis analysis and affinity binding measurements showed that the C-terminal segment of 4E-BPs is auxiliary for binding to eIF4E (25, 26). More recently, a conserved ⁷⁹PGVTS/T⁸³ motif found in the C terminus of 4E-BPs was shown to enhance its binding affinity to eIF4E from micromolar to nanomolar range (27, 28), revealing that 4E-BP1 has, in fact, a bipartite binding interface with eIF4E.Our group has identified a small-molecule inhibitor, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), which specifically disrupts association of eIF4G-derived peptides with eIF4E but stabilizes the eIF4E/4E-BP1 interaction (29). 4EGI-1 is of particular interest because it inhibits cap-dependent translation, is active against numerous cancer cell lines, and reduces growth of human cancer xenografts in vivo (29–31). Its effect is partially explained by the recent crystal structure of an eIF4E/4EGI-1 complex, in which the inhibitor binds to a site located remotely from the YXXXXLΦ binding interface, suggesting that it allosterically represses translation initiation (32). However, the mechanism by which 4EGI-1 stabilized 4E-BP1 binding remains unclear.In this study, we describe a crystal structure of eIF4E bound to a 35-residue fragment of 4E-BP1. This fragment comprises the consensus-binding motif and also a proline-turn-helix segment containing two phosphorylation sites (S₆₅ and T₇₀) followed by a loop of irregular structure. We find that the C-terminal loop of 4E-BP1 partially overlaps with the binding site of 4EGI-1, which enables us to understand the molecular mechanism through which 4EGI-1 inhibits translation initiation: by dissociating eIF4G from eIF4E but also stabilizing the interaction between eIF4E and the unphosphorylated form of 4E-BP1. We further find that the C-terminal loop of 4E-BP1 is required to inhibit cap-dependent translation and mediates cell cycle arrest in mammalian cells.     

慢性乙型肝炎患者血清乙型肝炎病毒表面大蛋白水平检测意义探讨

目的探讨慢性乙型肝炎、乙型肝炎肝硬化和原发性肝癌患者血清乙型肝炎病毒表面大蛋白（HBV-LP） 水平差异及意义。方法选取2014年8月~2015 年12月我院诊治的慢性乙型肝炎患者508例,乙型肝炎肝硬化患者74例,原发性肝癌患者29例,采用ELISA 法检测血清HBV-LP水平,采用FQ-PCR法检测血清HBV DNA水平。结果慢性乙型肝炎、乙型肝炎肝硬化和原发性肝癌患者血清HBV-LP阳性率分别为77.2%、82.4%和89.7%,血清HBV DNA阳性率分别为78.9%、83.8%和93.1%;3组血清HBV-LP水平分别为（9.78±4.25）μg/L、（17.24±8.63）μg/L和（38.65±19.38）μg/L,差异有统计学意义（P<0.05）。结论HBV-LP是乙型肝炎病毒感染者血清重要的标记物,与血清HBV DNA存在某种相关性,其检测的意义值得探讨。     

Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes

The core protein of hepatitis C virus (HCV) is believed to form the capsid shell of virus particles. Maturation of the protein is achieved through cleavage by host cell proteases to give a product of 21 000 MW, which is found in tissue culture systems and sera from infected individuals. However, efficient propagation of the virus is not possible at present in tissue culture. Hence, studies have focused on the properties of the core protein and its possible role in pathologies associated with HCV infection. This review describes key features of the polypeptide and the status of current knowledge on its ability to influence several cellular processes.     

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein–protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK–ACP complexes. Because transient enzyme–ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK–ACP complexes, allowing the determination of the crystal structure of the VinK–VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK–VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.Polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of various polyketide natural products (1). Bacterial modular PKSs comprise several catalytic modules that are each responsible for a single round of the polyketide chain elongation reaction. Each module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain. The AT domain recognizes a specific acyl building block and catalyzes its transfer reaction onto the 4′-phosphopantetheine arm of the ACP. KS extends the polyketide chain by condensing the resulting ACP-bound building blocks with the elongated acyl–ACPs. Although standard modular PKSs contain AT domains in their modules, some modular PKSs lack AT domains in each module and instead receive their acyl building blocks by standalone trans-acting ATs (2).The selection of the starter unit is generally governed by the substrate specificity of the AT domain in the loading module (1). In some modular PKS systems, a didomain-type loading module comprising a loading AT domain and an ACP domain selects an acyl starter building block such as an acetate unit to generate an acyl–ACP intermediate, which is transferred to the downstream extension module for polyketide chain elongation. Alternatively, in a KS_Q-type loading module consisting of three domains, the AT domain selects an α-carboxyacyl substrate such as a malonyl group, and the KS_Q domain subsequently catalyzes its decarboxylation to construct an acyl–ACP thioester. For polyketide chain elongation, the AT domain of the extension module generally recognizes a specific α-carboxyacyl–CoA as an extender building block (3). Malonyl– and methylmalonyl–CoA are commonly used as extender building blocks in biosynthetic pathways. Some ATs were reported to recognize ACP-bound substrates such as methoxymalonyl–, hydroxymalonyl–, and aminomalonyl–ACP (3, 4). Thus, ATs are key determinants of building block specificity in polyketide biosynthesis and attractive targets to change the substrate specificity to obtain biologically active unnatural polyketide products (5). However, the substitution of an AT domain by a homologous AT domain possessing different substrate specificity resulted in reduced or abolished production of polyketide analogs in many cases, probably because of disruption of proper protein–protein interactions or the inability of downstream modules to process polyketide analogs (5, 6).The importance of protein–protein interaction between AT and ACP during the acyltransfer reaction was proposed in previous studies (7, 8). Wong et al. described that AT recognizes its cognate ACP from other ACPs through protein–protein interactions (7). Proper AT–ACP interactions are believed to be essential for kinetically efficient polyketide chain elongation. However, the mechanism of ACP recognition is not well understood because isolated AT structures provide no detailed information on the AT–ACP interactions (9). Structural determination of the AT–ACP complex is necessary for the complete understanding of the basis of ACP recognition for the acyltransfer reaction.Macrolactam antibiotics are an important class of macrocyclic polyketides, and most contain a unique β-amino acid starter unit in their polyketide skeletons (10). Vicenistatin, produced by Streptomyces halstedii HC34, possesses a 3-aminoisobutyrate unit at the starter position of the polyketide backbone (11). This starter unit is biosynthesized from l-glutamate via (2S,3S)-3-methylaspartate, which is initially transferred onto the standalone ACP VinL by the adenylation enzyme VinN (12, 13). After decarboxylation, the resulting 3-aminoisobutyrate unit is aminoacylated with l-alanine to give dipeptidyl–VinL by another adenylation enzyme VinM. Then, the dipeptidyl moiety is transferred from VinL to the ACP domain (VinP1LdACP) of the VinP1PKS loading module by the trans-acting AT VinK (Fig. 1). These β-amino acid carrying enzymes are conserved in various macrolactam polyketide biosynthetic gene clusters, suggesting that β-amino acid starter units are loaded to PKS through the same mechanism in their biosynthesis (10).Open in a separate windowFig. 1.Biosynthetic pathway of vicenistatin, including the VinK reaction. The 3-aminoisobutyrate unit is shown in red.During the dipeptide transfer reaction from VinL to VinP1LdACP, VinK is supposed to recognize the VinL region as well as the dipeptidyl moiety to overcome the kinetic disadvantage of the diffusion-controlled limit. Additionally, VinK should distinguish VinP1LdACP as an acyl acceptor from other ACPs. Thus, we assume that the specific protein–protein interaction between VinK and two ACPs is important for the reaction. However, the origins of ACP selectivity cannot be predicted from the amino acid sequence of VinK. In this study, we carried out mutational and structural studies on VinK to clarify how VinK recognizes ACPs. The covalent VinK–VinL complex structure allows, to our knowledge, the first visualization of the interactions between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.     

Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein

Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.Pepducins represent a class of molecules that regulate the activity of G protein-coupled receptors (GPCRs). These lipid-modified peptides are derived from the amino acid sequences of one of the four intracellular loops of a target GPCR (1). Although the precise mode of action is not completely understood, it is believed that pepducins bind to their target receptors and allosterically modulate their signaling activity (2, 3). Pepducins have been identified for several GPCRs, including the protease-activated receptors PAR1 (1, 4–10), PAR2 (1, 11–13), and PAR4 (6, 9), the formyl peptide receptor-2 (14), the melanocortin type 4 receptor (1), the sphingosine 1-phosphate receptor 3 (15), and the C-X-C chemokine receptor type 1, 2 (CXCR1, CXCR2) (16), and 4 (CXCR4) (2, 17, 18). They have been found to act as allosteric agonists as well as negative or positive allosteric modulators. However, in most cases, their activity was assessed for only one or a few signaling pathways engaged by the receptors.One of the receptors for which pepducins were developed is CXCR4. This receptor, expressed in many tissues including hematopoietic and circulating cells, is a coreceptor for the entry of HIV (19, 20) and controls many physiological functions, including cell migration, mobilization, and retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem cells, as well as progenitor cells (HSPCs) in the bone marrow niche (21–23). It has also been found to play an important role in tumor progression, angiogenesis, and metastasis of a variety of cancers (24–26). A CXCR4 antagonist, AMD-3100 (Mozobil), is used to mobilize HSPCs from the bone marrow for transplantation of leukemic patients (27).A pepducin derived from the first intracellular loop of CXCR4 (ATI-2341) was found to be an allosteric agonist on the chemotactic response elicited on a human T-lymphoblastic leukemia cell line (CCRF-CEM cells) that endogenously expresses CXCR4 (18). This activity of ATI-2341 was confirmed in fresh human PMNs as well as in vivo for their ability to promote the mobilization of PMNs and HSPCs in the peripheral circulation of both mice and monkeys (18). Of note, unlike the clinically used CXCR4 antagonist AMD-3100 and stromal cell-derived factor-1α (SDF-1; also known as CXCL12) that promote the mobilization of lymphocytes in addition to PMNs and HSPCs (18, 28–30), ATI-2341 was without effect on the mobilization of lymphocytes (18), indicating that ATI-2341 may display functional selectivity.When assessing the effect of ATI-2341 on the signaling activity of CXCR4, the pepducin was found to be an allosteric agonist, activating the inhibitory heterotrimeric G protein (Gi) to promote inhibition of cAMP production and induce calcium mobilization (18, 31). In recent years, many GPCRs have been shown to engage in promiscuous signaling activities involving more than one G protein subtype as well as G protein-independent signaling. More importantly, it was found that different ligands can selectively couple to a subset of the signaling pathways that can be engaged by a receptor. In some cases, a given ligand can even have opposite efficacies on two different signaling pathways: a concept known as “ligand-biased signaling” or “functional selectivity” (32–36). In addition to its coupling to Gi, CXCR4 has also been found to signal through the engagement and activation of G13 (37, 38) and β-arrestin2 (39, 40), both pathways being proposed to contribute to the chemotactic responses. These observations raise the question of whether the CXCR4-selective pepducin, ATI-2341, is an allosteric agonist on all of the signaling pathways identified for the chemokine receptor or whether it could show bias toward selective signaling pathways and thus be functionally selective.To determine whether pepducins can display functional selectivity on CXCR4 signaling at the molecular level, we took advantage of bioluminescence resonance energy transfer (BRET)-based assays that allow the direct monitoring of the engagement and activation of proximal signaling effectors. More specifically, we compared the ability of ATI-2341 and the natural agonist of the receptor, SDF-1, to promote the engagement/activation of three Gi family members (Gi₁, Gi₂, and Gi₃), G13, β-arrestin1, and β-arrestin2. We found that, whereas SDF-1 promotes the engagement of all of the signaling effectors, ATI-2341 selectively led to the functional engagement of the Gi family members and had no effect on G13 or the two β-arrestins. The lack of recruitment of β-arrestins results from the poor recruitment of G protein-coupled receptor kinases (GRKs) to the receptor because, in contrast to SDF-1 that stimulates the phosphorylation of CXCR4 by protein kinase C (PKC), GRK2/3, and GRK6, ATI-2341 promotes effective PKC-dependent phosphorylation of the receptor but minimal GRK2/3 recruitment, as well as minimal GRK6-dependent phosphorylation.Taken together, our results demonstrate that the pepducin ATI-2341 is a functionally selective allosteric regulator of CXCR4 that activates Gi-dependent pathways without modulating G13 and β-arrestin pathways. These data indicate that ATI-2341 could have physiological actions that may differ from the natural ligand SDF-1 and the clinically used AMD-3100, raising the intriguing possibility that ATI-2341 may have distinct clinical properties. These findings also shed some light on the mechanism of action of pepducins and indicates that, similar to orthosteric ligands, these allosteric regulators can be functionally selective.     

Thermodynamic origins of protein folding,allostery, and capsid formation in the human hepatitis B virus core protein

HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry–mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.The “protein-folding problem” describes how a polypeptide sequence contains all the information needed for it to adopt a specific 3D structure spontaneously (1). The chemistry and thermodynamic code that causes proteins to fold also underpins protein–protein interactions, allostery, and supramolecular assembly. An emerging trend has been the study of model proteins free from kinetic traps, aggregation, or metal binding, features that can confound experimental execution and data interpretation (2, 3). Consequently, model proteins are small (typically <130 residues), soluble monomers with few proline or cysteine residues and no prosthetic groups (2, 3).Although model proteins have been instrumental in taking the field to its current zenith, there is a paucity of experimental insights into the conformational dynamics of larger, oligomeric proteins, especially those implicated in diseases (3). Such proteins usually have complex behavior refractory to detailed experimental studies. However, the connection between sequence, structure, dynamics, and allostery makes studies of larger proteins central to understanding biological function and aiding drug design (vide infra) (4). One such protein is HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), a major pathogen that kills 600,000 people annually (5). Although excellent vaccines exist, there are no effective cures for extant chronic infections (5, 6). In addition to capsid formation, HBc plays many essential roles in HBV replication (7–9), making it an attractive drug target (10–15).WT HBc is a 183-residue polypeptide comprising a structured capsid-forming region (residues 1–149; Fig. 1A) and a basic, nucleic acid-binding domain (residues 150–183) (16–18). The structured N-terminal region (hereafter HBc_1–149) spontaneously self-assembles in vitro and in vivo to form icosahedral capsid-like particles (CLPs) identical to nucleocapsids isolated from patient serum (19, 20). X-ray crystallography and cryo-EM have characterized the structure of HBc_1–149 within the context of CLPs, virions, and hexamers (16, 19–23). HBc homodimers comprise two structural domains (Fig. 1A): Helices α₃ and α₄ from opposing monomers pack together and form a disulfide-linked, four-helix bundle dimerization interface (visible as protrusions on the capsid exterior; Fig. 1B), whereas α₁, α₂, and α₅ pack together and around the base of the four-helix bundle to create the hydrophobic core of “contact” domains (19). Weak interdimer interactions between contact domains stabilize HBV capsids (19, 24) (Fig. 1B).Open in a separate windowFig. 1.HBc_1–149 dimer structure within HBV capsids. (A) Four-helix bundle dimerization interface (black) is flanked by contact domains (orange and red). Helices are numbered, and the N and C termini of one monomer are indicated. The disulfide link between C61 of each monomer is indicated (cyan). (B) Exterior surface of a T = 4 capsid HBc_1–149 (PDB ID code 1QGT) (19). Dimers around the threefold and fivefold axes are indicated in blue/green and purple/orange, respectively. (Inset) Interacting quasiequivalent HBc_1–149 dimers from the fivefold (purple and orange) and threefold (blue and green) axes are shown. Hydrophobic contacts between contact domains stabilize capsids. Residues that perturb capsid formation when mutated are indicated.Multiple studies show clearly that HBc has a very malleable structure, with this structural plasticity argued to be functionally important (22, 23). This hypothesis accords well with antivirals that modulate HBc structure (11–15, 22, 23). Studies of HBV capsid assembly have inferred the existence of assembly-active (HBc^Ass) and assembly-incompetent (HBc^Inc) HBc conformations (12, 13, 21, 24, 25). However, there are few detailed insights on the thermodynamic origins of structure, allostery, and dynamics for the dimeric HBc_1–149 protomer, where structural plasticity must originate. This arises from dimeric HBc_1–149 being very challenging to study in vitro (compared with the model proteins described above) because it is a 298-residue disulfide-linked homodimer (containing 6 cysteine and 24 proline residues) that aggregates aggressively and forms capsids.Here, we report detailed folding and stability studies of dimeric HBc_1–149. These show HBc_1–149 folds in a three-state transition with a populated, dimeric, α-helical intermediate. Of 29 “chemically conservative” mutants used to probe folding energetics (26), many had similar effects on the stability of the intermediate and native ensembles. The distribution of these mutations was consistent with the intermediate being stabilized by a significant native-like structure. However, some mutations destabilized the native state (N) much less than the intermediate state (I) relative to the denatured state (D), or significantly increased the free energy of unfolding (ΔG_D–N) relative to WT HBc_1–149. This suggests HBc_1–149 contains structural strain arising from frustrated contacts (27, 28). We found evidence of HBc_1–149 adopting multiple native conformers, where capsid assembly-competent conformers were less stable than those incapable of, or attenuated in, capsid formation. Frustrated regions likely contain structural gatekeepers that (28), when mutated, subtly tuned the folding energy landscape and altered capsid assembly. The presence of multiple native conformations and frustrated regions may explain the origins of allostery reported for HBc. Frustration is likely an evolved tradeoff that balances the conflicting requirements of HBc folding with allosteric regulation of native structure, capsid formation, and diverse functions of different conformers (29). The ability to trap HBc conformers thermodynamically by mutation and ablate capsid formation provides a proof of principle for designing antivirals that elicit similar effects.     

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-α therapy ( n =37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli

Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-Å resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein–protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.     

Acute exacerbation of hepatitis due to reactivation of hepatitis B virus with mutations in the core region after chemotherapy for malignant lymphoma

A 43-year-old Japanese man who was positive for hepatitis B surface (HBs) antigen and HB e antibody, underwent chemotherapy for non-Hodgkin’s lymphoma. After the chemotherapy he suffered from acute exacerbation of hepatitis because of reactivation of HBV. Recovery was achieved with interferon-α, glucagon-insulin therapy, and plasma exchange. Mutations were detected in codons 97, 100, 129, and 131 of the core region of HBV. The peptide encoded from the core region including such mutations possibly had greater antigenicity to induce cytotoxic T cell activity in the host. Core region mutations may be a crucial cause of the acute exacerbation of hepatitis B seen after chemotherapy.