云南切梢小蠹气味结合蛋白的同源模建

【目的】为了研究云南切梢小蠹Tomicus yunnanensis气味结合蛋白(Odorant binding proteins,OBPs)参与其嗅觉识别过程的机理奠定基础,本文对云南切梢小蠹OBP进行了同源模建及评价分析。【方法】采用同源模建方法构建了云南切梢小蠹OBP的三维结构,并利用Procheck、Verify_3D和ERRAT等方法评估了构建模型的可靠性。【结果】对优化后的模建蛋白的结果分析发现,序列中有6个保守的半胱氨酸位点,具有气味结合蛋白的典型特征,并且Procheck、Verify_3D和ERRAT对模型的评价分数较高,结构具有很高的可能性。【结论】基于已知的同源蛋白结构信息和模建蛋白的一级序列信息,预测序列已知但结构未知的蛋白空间结构,为今后研究该云南切梢小蠹气味结合蛋白的功能结构域奠定基础。     

HLA—DRα＊0101、DRβ1＊04051和DRβ1＊07011的分子克隆、测序及空间结构模建

目的：Y国探索HLA（human leukocyte antigens,HLA)不同分子的空间结构差异与功能之间的关系以及结构差异能否对预测造血干细胞移植GVHD（graft-versus-host disease,GVHD)的发生奠定基础。方法：提取人外周血总RNA,RT-PCR扩增HLA——DR等位基因全序,连接、转化大肠杆菌后并测序,将第二外显子氨基酸序列输入微机,通过INTER-NET传送至SWISS-MODELLING蛋白质空间结构模建服务器进行结构模建,结果：本文反转录了2例健康无血缘关系个体的HLA-DR位点基因全序,测序结果与HLA-DRα*0101、DRβ1*04051和DRβ1*07011的公布序列完全一致。对后两者的蛋白质空间结构模建发现它们在空间结构上存在显著差异。结论：对HLA基因位点进行全长测序可以明确判断基因型别;空间结构模建可以直观地辨别HLA不同舂子在空间结构上的差异。这为研究HLA结构与功能的关系以及HLA不完全相合的造血干细胞移植后预测GVHD的研究提供实验基础。     

利用序列比较寻找胰岛素基因表达启动区与DNA结合蛋白的结合位点

NDFl、IPFl和HNF4是与胰岛素基因表达有关的DNA结合蛋白,通过比较SWISSPROT蛋白质数据库中人类、小鼠、大鼠这三种核蛋白氨基酸一级序列、模体和结构域,发现其结构十分相似,根据蛋白质结构和功能的关系,推测这些DNA结合蛋白与胰岛素基因结合的核苷酸序列相似;从GenBanl(核酸数据库中获得人类、小鼠、大鼠胰岛素DNA序列,用ClustalW比较三者Promoter区的核苷酸序列,显示有一段核苷酸序列较为相似,同时搜索TRANSFAC基因转录数据库中NDFl、IPFl和NHF4蛋白核苷酸结合位点,发现核酸比对保守的部分序列与TRANSFAC数据库中这三个转录因子的DNA结合位点一致,另外一些核酸保守序列可能为其他未知DNA结合蛋白的结合位点。这种核酸序列比对设计为分子生物学实验寻找和验证胰岛素DNA结合蛋白与核苷酸的结合位点提供了简单而实用的方法。     

源于红树林的新型脂肪酶Lip906生物信息学分析

本研究利用生物信息学在线软件对脂肪酶Lip906的二级结构和模体信息进行预测,同时对其三级结构进行同源建模和模建结果质量评价,预测该蛋白质的活性位点信息,旨在从蛋白质序列特征和分子结构水平理解其在酯类水解过程中的作用。结果表明,模建的Lip906蛋白结构品质较高,具有7段α-螺旋和2组β-折叠结构,是一个典型的α/β类蛋白,表面呈弱负电势分布;Lip906蛋白具有5个不同模体,可能参与不同生化反应或执行不同的功能。这些研究结果对理解Lip906蛋白功能以及配基结合位点定位非常重要,也为脂肪酶Lip906的突变设计提供了理论基础。     

肺炎链球菌双组份系统中的组氨酸激酶(YycG)的同源模建与分析

对肺炎链球菌双组份系统中的组氨酸激酶YycG进行同源模建, 并分析其与底物ADP的相互作用, 为寻找特异性的激酶抑制剂提供了理论依据。采用同源模建的方法构建YycG蛋白的三维结构, 并用ProCheck、Profile_3D软件对此结构模型的合理性进行验证; 用Autodock4.0软件将结构模型与ADP进行自动对接, 分析二者之间的相互作用。序列比对结果显示肺炎链球菌YycG蛋白与Thermotoga maritima X-ray晶体结构序列的同一性达33%; YycG模建后的结构与模板能很好的叠合; 在活性口袋处的保守的氨基酸残基Asn145、Asn149、Lys152以及口袋内部的疏水残基在结合、水解底物ADP的过程中发挥重要作用。组氨酸激酶YycG的模建合理, 该结构模型可作为设计抗菌药的研究起点。     

肝细胞生长因子Kringle的三维结构模建及与功能的关系

利用同源模建的方法模拟得到了肝细胞生长因子4个Kringle域的三维结构。结果表明,HGFKringle与纤溶酶原Kringle的氨基酸序列具有较高的同源性,其功能区附近的序列比较保守。HGF的Kringlel和3与其它具有Lys结合功能的Kringle相比,功能区的残基发生了变化,可能丧失了结合Lys的功能,而2和4仍具有一定的该功能。根据Kringle 1的模建结构,推测该Kringle功能区的结构为一个通道,该通道的底部和一侧有部分疏水残基,同时两侧还分布着少量酸性或碱性残基,该通道可能具有结合特定肽链的功能,从而与Kringle 2一起实现HGF与受体结合的作用。     

Profile-profile比对方法用于发现远距离同源模板

基于模板的模建方法是蛋白质结构预测领域中最为准确有效的方法,该类方法的成功与否对模板质量的要求较高。为待预测序列找寻合适的模板,本文提出了一种profile-profile比对的方法将查询序列同模板库中的已知结构蛋白进行比对,然后根据比对结果的Z-score得分高低顺序挑选出合适的模板。结果表明:本文的profile-profile比对方法在测试集上的性能明显优于PSI-BLAST,相比PSI-BLAST在测试集上的准确度提高了约14.3%,配对t检验的结果表明准确度的提高具有统计显著性。从而得出如下结论:本文的profile-profile比对方法可以用于为序列相似性较低的待预测序列搜索远距离同源模板,并用于指导后续的三级结构预测。     

裂殖壶菌EST文库cdc2基因的筛选与分析

利用已建立的裂殖壹菌(Schizochytrium sp FJU-512)EST文库,通过序列比对分析发现p34cdc2相似基因的3′端序列,分离含该EST序列的质粒ROCHA2004,以T7引物进行反向测序,获得p34cdc2相似基因的全长序列,结合NCBI的ORF finder以及Blastx程序获得p34cdc2蛋白的氨基酸序列;利用同源模建方法构建了该蛋白的三维空间结构,并预测其功能结构域;对该蛋白进行系统发育分析,构建进化树,结果显示该蛋白同小麦(Triticum aestivum)的p34cdc2序列亲缘关系最近。     

Myostatin三维结构模建及分子进化分析

Myostatin(MST)为肌肉生长负调节因子,其功能受抑制可导致肌肉量增加.对MST核酸序列进行序列比对,构建进化树;采用同源模建方法首次模建MST成熟肽生物活性二聚体的四级结构,并预测MST与其受体ActRIIB的相互作用模式.进化树将肌肉生长抑制素基因(MSTN)分成4个亚家族:哺乳动物MSTN,鸟类MSTN以及鱼类MSTN 1和2.MST受纯化选择作用,在不同物种的直系同源基因具有较高的刚源性,其中哺乳动物、鸟类MST C端活性肽氨基酸序列高度保守.表明哺乳动物、鸟类MST的结构、功能类似,且信号传导路径可能一致;而鱼类MST的调控机制可能存在较大差异.MST结构及其表面静电势和疏水氨基酸分布表明静电力和疏水相互作用在MST与其受体结合过程中可能起到十分重要的作用.     

R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据.采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck 验证结构模型的合理性.使用Suflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用.序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71％.Deh-R模建后的结构与模板很好的吻合.模型比对分析DehI-R中参与催化的残基,除Asn2.03外大部分都比较保守.分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是值点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Aan具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应.利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln.突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用.     

Characterization of human inducible costimulator ligand expression and function

The inducible costimulator (ICOS) is the newest member of the CD28/CD152 receptor family involved in regulating T cell activation. We constructed a soluble-Ig fusion protein of the extracellular domain of human ICOS and used it as a probe to characterize expression patterns of the ICOS ligand (ICOSL). ICOSIg did not bind to CD80- or CD86-transfected Chinese hamster ovary cell lines, demonstrating that ICOSL is distinct from those ligands identified for CD28/CD152. ICOSIg showed selective binding to monocytic and B cell lines, whereas binding was undetectable on unstimulated monocytes and peripheral blood T and B cells. Expression of ICOSL was induced on monocytes after integrin-dependent plastic adhesion. Pretreatment of monocytes with mAb to the beta2-integrin subunit CD18 decreased adhesion and abolished ICOSL up-regulation but had no effect on CD80/86 (CD152 ligand (CD152L)) expression. Both ICOSL and CD152L were up-regulated on monocytes by IFN-gamma but by distinct signaling pathways. Unlike CD152L expression, ICOSL expression did not change when monocytes were differentiated into dendritic cells (DCs) or after DCs were induced to mature by LPS, TNF-alpha, or CD40 ligation. Addition of ICOSIg to allogeneic MLRs between DCs and T cells reduced T cell proliferative responses but did so less efficiently than CTLA4Ig (CD152Ig) did. Similarly, ICOSIg also blocked Ag-specific T cell proliferation to tetanus toxoid. Thus, ICOSL, like CD80/86, is expressed on activated monocytes and dendritic cells but is regulated differently and delivers distinct signals to T cells that can be specifically inhibited by ICOSIg.     

Modeling and docking the endothelin G-protein-coupled receptor

A model of the endothelin G-protein-coupled receptor (ET(A)) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane alpha-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was modeled as a three-helix domain based on its homology with a hydrolase protein. To test the model, the FTDOCK algorithm was used to predict the ligand-binding site for the crystal structure of human endothelin. The site of docking is consistent with mutational and biochemical data. The principal sites of interaction in the endothelin ligand all lie on one face of a helix that has been implicated by structure-activity relationship studies as being essential for binding. As further support for the model, attempts to dock bigET, an inactive precursor to endothelin that does not bind to the receptor, found no sites for tight binding. The model of the receptor-ligand complex produced forms a basis for rational drug design of agonists and antagonists for this G-protein-coupled receptor.     

Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance

Gram-negative bacteria have evolved mechanisms to resist the bactericidal action of cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. The strategy involves the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A in their outer membrane. ArnA is a key enzyme in the Ara4N-lipid A modification pathway. It is a bifunctional enzyme catalyzing (1) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to the UDP-4' '-ketopentose [UDP-beta-(l-threo-pentapyranosyl-4' '-ulose] and (2) the N-10-formyltetrahydrofolate-dependent formylation of UDP-Ara4N. Here we demonstrate that the transformylase activity of the Escherichia coli ArnA is contained in its 300 N-terminal residues. We designate it the ArnA transformylase domain and describe its crystal structure solved to 1.7 A resolution. The enzyme adopts a bilobal structure with an N-terminal Rossmann fold domain containing the N-10-formyltetrahydrofolate binding site and a C-terminal subdomain resembling an OB fold. Sequence and structure conservation around the active site of ArnA transformylase and other N-10-formyltetrahydrofolate-utilizing enzymes suggests that the HxSLLPxxxG motif can be used to identify enzymes that belong to this family. Binding of an N-10-formyltetrahydrofolate analogue was modeled into the structure of ArnA based on its similarity with glycinamide ribonucleotide formyltransferase. We also propose a mechanism for the transformylation reaction catalyzed by ArnA involving residues N(102), H(104), and D(140). Supporting this hypothesis, point mutation of any of these residues abolishes activity.     

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed.     

Crystal structure of a methyltetrahydrofolate- and corrinoid-dependent methyltransferase

BACKGROUND: Methyltetrahydrofolate, corrinoid iron-sulfur protein methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation. It transfers the N5-methyl group from methyltetrahydrofolate (CH3-H4folate) to a cob(I)amide center in another protein, the corrinoid iron-sulfur protein. MeTr is a member of a family of proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or -sarcino)pterin. We report the first structure of a protein in this family. RESULTS: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 A resolution using multiwavelength anomalous diffraction methods. The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold. The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity. This homology permitted the methyl-H4folate binding site to be modeled. The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the methyltransferases and dihydropteroate synthetases. The most significant structural difference between these enzymes is in a loop structure above the active site. It is quite open in MeTr, where it can be modeled as the cobalamin binding site. CONCLUSIONS: The MeTr structure consists of a TIM barrel that embeds methyl-H4folate and cobamide. All related methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site. The observed structure is consistent with either a 'front' (N5) or 'back' (C8a) side protonation of CH3-H4folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).     

The X-ray crystallographic structure of Escherichia coli branching enzyme

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.     

Predicted three-dimensional structure of the protease inhibitor domain of the Alzheimer's disease beta-amyloid precursor

Alzheimer's disease is characterized by the deposition of amyloid beta-protein as plaques and tangles in the brains of its victims. The amyloid precursor can be expressed with or without the inclusion of a protease inhibitor domain, the potential role of which in amyloidogenesis has prompted the generation of a model of its three-dimensional structure based on the known structure of a related inhibitor. The model structure predicts that the mutated residues are almost entirely on the surface of the inhibitor domain, while conserved residues constitute the hydrophobic core. In addition, several pairs of structurally complementary, or concerted, mutations are seen. These structural features provide strong evidence for the validity of the modeled structure, and it is suggested that the presence of complementary mutations may be used as a criterion for evaluating protein structures built by homology, in addition to the (spatial) location of the mutations. The terminal residues delimiting the domain are among those furthest from the protease binding site and are in close proximity to one another, thus suggesting the ability of the domain to function as a structural cassette within the context of a larger protein. The electrostatic potentials of the inhibitor and of the related bovine pancreatic trypsin inhibitor reveal how two inhibitors with very different net charges can bind with approximately the same binding constant to trypsin and suggest a mutation of trypsin that might selectively enhance the binding of the amyloid inhibitor domain. The model provides a structural basis for understanding the functional roles of residues in the domain and for designing simpler molecules to test as pharmacologic agents for intervention in Alzheimer's disease.     

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.     

Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli. Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP. Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled. The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues. The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP. The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues. The model also is consistent with cAMP binding in the syn conformation in both sites. Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts. This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.