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。     

t-PA K1区的同源模建及Kringle区与赖氨酸相互作用的研究

利用同源模建方法预测了t-PAK1区的三维结构。通过结构叠合确定了t-PAK1、K2区,纤溶酶原K1、K4区及UKK区的赖氨酸结合口袋。静电势计算及疏水性分析表明,在t-PAK2区以及纤溶酶原K1、K4区与纤维蛋白裸露的赖氨酸之间存在明显的静电势互补和疏水面契合。确定了影响Kringle区结合口袋与赖氨酸亲和的重要氨基酸,分析了t-PAK1区、UKK区不能结合赖氨酸的原因,由此设计了具有赖氨酸亲和力的新型t-PAK1区及UKK区突变体。利用模拟残基突变技术预测了突变体的结构变化,分析了突变后t-PAK1区及UKK区与赖氨酸亲和力的变化,初步在理论上肯定了设计方案的合理性。     

融合蛋白ICOSIg的三维结构模建的研究

目的:利用结构相似性的序列比对来模建ICOSIg的三维结构,分析其可能的结合位点,为改造ICOSIg的突变体,提高其结合活性提供理论基础。方法:利用生物信息学手段分析ICOS所属CD28家族各成员分子的结构域,通过基于结构相似的序列比对,以空间结构已经得到解析的CTLA4为模板,利用同源模建的方法,模建ICOS膜外区的空间结构。进一步地以人IgG2和CTLA4为模板,模建了ICOSIg全长的空间结构。在此基础上,结合氨基酸特性,分析其可能的功能位点。结果:FDPPPF及KTKGSGN基序可能是ICOSIg的功能结合位点。结论:模建了ICOSIg的空间结构,分析了其可能结合位点,为突变ICOSIg提高其亲和力提供了线索。     

植物Rubisco活性中心的模拟分析

通过对与不同配基结合的植物Rubisco复合物结构的重叠比较分析 ,发现Rubisco的活性差异是由其中一段Loop6环序列所造成的 ;金属离子与活性中心的结合会造成活性中心巨大的构象变化 .进一步用SwissPDBViewer软件模拟不同配基的植物Rubisco活性中心与此Loop环的氢键相互作用 .结果表明 ,有 3个Lys残基Lys2 0 1、Lys334、Lys175与Rubisco是否处于活性状态密切相关 ,这些残基的结构变化对分子设计可能有重要的参考价值     

模式匹配分析方法检索免疫球蛋白样功能区

运用计算机进行核酸和蛋白质的序列分析是分子生物学研究的一个较新发展,这项技术已越来越多地用于研究大量积累的序列数据。蛋白质功能区是蛋白质分子中能独立折叠成具有一定结构并执行特定功能的结构域,所有具有同一类功能区的分子统称为一个蛋白质的超族(protein superfamily)。本文通过对免疫球蛋白(Ig)超族及其功能区序列所进行的分析,建立了一种根据功能区之保守片段残基组成的模式匹配分析检索蛋白质功能区的方法,它先根据多序列的对准比较确定某一类功能区之保守片段,再对已知的保守片段各位置上氨基酸残基组成进行统计分析,然后根据与统计数值相匹配的方法,计算待检序列残基组成的统计学意义,由此确定功能区的存在。该方法的优点在于它不仅可以检出已知的具有某一类功能区的分子,而且还可能发现新的具有该功能区的分子,从而推测后者的功能。     

白色念珠菌羊毛甾醇14α-去甲基化酶三维结构分子模型研究

基于四种原核细胞色素Ｐ４５０晶体蛋白Ｐ４５０ＢＭ３、Ｐ４５０ｃａｍ、Ｐ４５０ｔｅｒｐ、Ｐ４５０ｅｒｙＦ模建白色念珠菌羊毛甾醇１４α－去甲基化酶三维结构。序列匹配采用四种晶体结构比较结果基础之上提出的细胞色素Ｐ４５０超家族蛋白基于结构知识的序列匹配方法。以Ｐ４５０ＢＭ３晶体结构坐标模建目标蛋白结构保守区主链结构,结构保守区侧链构象来源于四种晶体蛋白与模建蛋白对应残基同源性得分最高残基构象。模建结果用分子力学和分子动力学进行结构优化,模建结果蛋白采用Ｐｒｏｆｉｌｅ－３Ｄ图、Ｒａｍａｃｈａｎｄｒａｎ图和疏水图分析确证结构的合理性。并根据模型推测与血红素辅基相互作用的残基、与氧化还原偶联蛋白作用和参与电子传递的残基、底物进出通道和活性位点的残基。这些研究结果为定点突变研究、抗多肽抗体结合实验等提供理论依据,为高效低毒抗真菌药物合理设计提供靶标。     

具重复Kringle结构分子的缺失体和嵌合体的构建方法

确定肝细胞生长因子(HGF)和巨噬细胞刺激蛋白(MSP)各4个Kringle结构的共有序列并设计公用引物,此公用引物配合HGF和MSP上、下游引物,利用DNA聚合酶链式反应(PCR)技术,可在一次PCR反应中扩增出多条片段,以不同的组合方式将这些片段两两连接,得到了缺失不同Kringle的缺失体和两种分子间不同区域的缺失型嵌合体,从而为HGF和MSP结构与功能的研究奠定了基础。此方法可普遍适用于含有两个或两个以上Kringle分子的缺失体与嵌合体的构建。克隆过程中对存在引物酶切位点的cDNA片段的克隆方法的建立,为同类问题的解决提供了可靠的方法。     

鼠源单克隆抗体HAb25可变区的CDR-移植法人源化计算机辅助设计

目前鼠源抗体人源化是克服其免疫原性的主要手段。在结构模建基础上的计算机分析为抗体人源化设计提供了必不可少的辅助。本设计首先对肝细胞癌特异性抗体HAb25可变区进行了同源模建,然后分析决定CDR初始构象的可能残基,包括正则结构关键残基、CDR可接触残基、界面残基、包埋残基,以及与免疫识别密切相关的表面残基,综合考虑并结合多重序列比较,参照人源化模板,提出了人源化替代的方案。     

HPO结构与受体结合功能关系的初步研究

肝细胞生成素(HPO)是一种新型细胞因子,在肝再生过程中具有重要的调控作用,研究发现在肝源细胞表面有其特异性受体。为了研究HPO结构与其受体结合功能的关系,构建并表达了HPO及其系列缺失体,利用流式细胞术检测了HPO及其系列缺失体与受体的结合能力。结果表明,在HPO的序列中可能有多个受体结合功能区,其N端的前10位氨基酸残基和C端的第11—20位氨基酸残基对于其受体结合能力是必需的,而C端前10位氨基酸残基对HPO与受体的结合有一定的抑制作用。此外HPO结构的完整性对于其受体结合功能也是必需的。     

具重复Kringle结构分子系列缺失体和嵌合体的构建方法及验证

确定肝细胞生长因子 (HGF)、巨噬细胞刺激蛋白 (MSP)各 4个Kringle结构的共有序列并以此为基础设计公用引物 ,利用此公用引物可扩增出含不同数目Kringle的系列片段 ;以不同的组合方式连接 ,得到了系列缺失体和嵌合体 ;测序结果表明公用引物的简并性及所扩增序列与理论预期一致 .对以此法所构建的HGF/MSPK1缺失体表达后进行活性测定 ,提出MSP的K1是重要的功能决定区 .此外 ,提出了对内部存在引物酶切位点的cDNA片段的克隆策略 .     

Kringle 5 of human plasminogen carries a benzamidine-binding site

Affinity of plasminogen fragments for p-aminobenzamidine-Sepharose was investigated to localize the benzamidine-binding site(s) of the protein. i/ Of the elastase fragments of plasminogen only miniplasminogen (kringle 5 plus light chain) was bound to the column. Kringle 1+2+3 and kringle 4, which carry the lysine-binding sites, were not adsorbed, proving that the lysine-and benzamidine-binding sites are on different domains of the protein. ii/ Light chain was bound to the column even if the primary benzamidine-binding site was covalently blocked, indicating that the protease part of plasmin has a second benzamidine-binding site. iii/ Kringle 5 also binds to the affinity column: the presence of a binding site on kringle 5 raises the possibility that this structure may take part in the interactions of plasminogen with other proteins.     

A 1H-NMR study of isolated domains from human plasminogen. Structural homology between kringles 1 and 4

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr approximately equal to 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H-NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p-benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysine-binding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl-region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two-dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at -1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H-NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine-binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine-Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine-binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.     

Deriving the generic structure of the fibronectin type II domain from the prothrombin Kringle 1 crystal structure.

The proposed homology between the fibronectin type II domain and the Kringle domains of blood clotting and fibrinolytic proteins has been examined in three dimensions by substituting the type II sequence into the bovine prothrombin Kringle 1 tertiary structure, determined by X-ray crystallographical methods at 3.8 A. Structural substitution of aligned amino acids of the type II domains and the Kringle produces a compact chain fold and deletions and insertions in the type II sequence are accommodated within the modelled structure. This confirms the structural homology between the two domains and verifies the sequence alignment and common evolution of the type II and Kringle units. The two structures contain homologous hydrophobic cores, centered around the two disulphide bridges which link conserved beta-type strands. Gross differences between the two domains occur in exterior loops and potential functional sites in these regions of the type II structures as found in fibronectin, Factor XII and seminal fluid protein PDC-109 are proposed. We suggest that the domains evolved from a common ancestral protein comprising the hydrophobic core and disulphide arrangement which later diverged to bind different macromolecules through adaptation of the external loops.     

Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-LIKE gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.Key words: ClC channel, chloride channel, homology model     

Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.     

Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.     

台湾家白蚁内切葡聚糖酶活性中心氨基酸的饱和突变

对内切葡聚糖酶的功能改进一直是纤维素酶研究领域的焦点。本研究对台湾家白蚁内切葡聚糖酶(CfEG)的活性位点做了饱和突变。首先,以PDB数据库中高山象白蚁内切葡聚糖酶(NtEG)的三维结构(PDB id=1ks8)为模板,对CfEG进行三维结构同源建模,二者序列一致性高达79%。位于CfEG活性中心的D53、D56、E411,分别与NtEG的催化残基D54、D57、E412重合。用简并引物对CfEG的假定活性位点D53、D56、E411进行定点饱和突变。在位点D53、D56各筛选到羧甲基纤维素酶活有一定提高的突变子D53E、D56C,其中D56C的Km值减小为原始酶的三分之一。双突变子D53L/D56I的比活比原始酶提高了近2倍,同时Km值减小至原始酶的一半。而E411的饱和突变子库均没有活性,进一步将其替换为近似氨基酸的E411D、E411Q定点突变子也丧失了酶活。由突变结果可推断,位点E411为该酶行使功能的必需残基。     

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.