Structural Basis of Glycan Recognition in Globally Predominant Human P[8] Rotavirus

Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3 ?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.     

Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent,Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment

The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA’s fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA’s fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (～70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.     

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录-聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH-2vp7基因,连接到克隆载体pUCm-T,并对其基因序列进行了分析.WH-2与ADRV的同源性达98%,与印度加尔各达分离株CAL-1达92%,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58%,与WD653(牛)B组轮状病毒同源性为63%,与ATI(牛)同源性为61%.对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构.VP7蛋白是249个氨基酸组成,分子量为28.4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH-2与ADRV的同源性达99%,与CAL-1达95%,而IDIR仅为51%,说明了WH-2和ADRV的起源相同.此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据.     

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录—聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH—2vp7基因,连接到克隆载体pUCm—T,并对其基因序列进行了分析。WH—2与ADRV的同源性达98％,与印度加尔各达分离株CAL—1达92％,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58％,与WD653(牛)B组轮状病毒同源性为63％,与ATI(牛)同源性为61％。对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构。VP7蛋白是249个氨基酸组成,分子：量为28．4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH—2与ADRV的同源性达99％,与CAL—1达95％,而IDIR仅为51％,说明了WH—2和ADRV的起源相同。此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据。     

Structural Correlates of Rotavirus Cell Entry

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.     

Structural Basis for Activity Regulation and Substrate Preference of Clostridial Collagenases G,H, and T

Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.     

人轮状病毒HRB-02株的分离鉴定

从哈尔滨医科大学第二临床医学院发生腹泻患儿粪便中取得标本 ,用MA10 4细胞传代培养 ,传至第4代时 ,细胞出现明显病变 ;电镜观察见到典型的轮状病毒颗粒 ;其RNA电泳型为 4∶2∶3∶2型 ,属A群轮状病毒。对其蚀斑特性和血凝特性进行了初步研究 ,把这株轮状病毒地方株命名为HRB 0 2株。为研究该株轮状病毒的分子生物学特性 ,从而研制诊断性抗原和疫苗奠定基础。     

Structural Basis of Activation of Cys-Loop Receptors: the Extracellular–Transmembrane Interface as a Coupling Region

Cys-loop receptors mediate rapid transmission throughout the nervous system by converting a chemical signal into an electric one. They are pentameric proteins with an extracellular domain that carries the transmitter binding sites and a transmembrane region that forms the ion pore. Their essential function is to couple the binding of the agonist at the extracellular domain to the opening of the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50?Å to the gate is therefore central for the understanding of the receptor function. A step forward toward the identification of the structures involved in gating has been given by the recently elucidated high-resolution structures of Cys-loop receptors and related proteins. The extracellular–transmembrane interface has attracted attention because it is a structural transition zone where β-sheets from the extracellular domain merge with α-helices from the transmembrane domain. Within this zone, several regions form a network that relays structural changes from the binding site toward the pore, and therefore, this interface controls the beginning and duration of a synaptic response. In this review, the most recent findings on residues and pairwise interactions underlying channel gating are discussed, the main focus being on the extracellular–transmembrane interface.     

Structural Studies of G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) constitute the largest and the most physiologically important membrane protein family that recognizes a variety of environmental stimuli, and are drug targets in the treatment of numerous diseases. Recent progress on GPCR structural studies shed light on molecular mechanisms of GPCR ligand recognition, activation and allosteric modulation, as well as structural basis of GPCR dimerization. In this review, we will discuss the structural features of GPCRs and structural insights of different aspects of GPCR biological functions.     

蛋白质磷酸化作用的结构基础

蛋白质可逆磷酸化涉及到几乎所有细胞活动的调节.着重探讨了影响蛋白激酶作用专一性的几个因素和磷酸化影响蛋白质功能的结构基础及作用机制.     

A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.     

Conditional Knockout of NMDA Receptors in Dopamine Neurons Prevents Nicotine-Conditioned Place Preference

Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction.