组氨酸激酶(YycG)在筛选肺炎链球菌抑制剂中的应用

为了获得具有体外活性的肺炎链球菌组氨酸激酶YycG并利用其筛选寻找新的抑制剂。原核表达组氨酸激酶YycG的激酶功能域,经SDS-PAGE,Western blot鉴定及镍层析柱纯化后,采用激酶试剂盒检测其激酶活性;利用对其激酶活性的抑制作用从105种候选化合物中筛选有效的抑制剂,并通过实验验证抑制剂的抗菌作用。原核表达得到约35 kDa的目的蛋白激酶域片段YycG′,其纯度达 95 %,并具有体外水解ATP的激酶活性;利用其活性筛选得到数种不同抑制效果的小分子化合物,且体外验证具有较好的抑菌效果。通过肺炎链球菌组氨酸激酶YycG活性筛选找到的小分子抑制剂可为进一步研发与该菌相关的药物或消毒剂提供基础。     

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.     

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。     

来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究

立体选择性是2-卤代酸脱卤酶最重要的性质之一,但目前其手性识别过程尚不明确,对其进行研究和解析具有重要意义。以来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶dehDIV-R为模型,研究了R-2-卤代酸脱卤酶的手性识别过程。首先通过测定反应产物的构型,确定dehDIV-R催化底物为S_N2反应。通过Discovery Studio 3.0对dehDIV-R进行同源模建及底物分子对接,由对接结果和序列比对确定dehDIV-R立体选择性的关键位点Asn236,预测dehDIV-R的立体选择性与反应时底物到达反应位置的空间位阻密切相关。对dehDIV-R进行虚拟突变,将Asn236位点突变成具有不同空间位阻的残基Ala和Ser,并分别与底物分子进行分子对接,预测突变酶的立体选择性。根据预测结果,对Asn236氨基酸残基进行定点突变,发现在Asn236突变为Ala后的A1酶显示出对RS底物的活力;在Asn236突变为Ser后的S1酶显示出与原始酶相反的立体选择性,实现了立体选择性的反转。与模型的预测结果相符,证明了模型的合理性。     

Furin／kexin蛋白质前体加工酶抑制剂的理性再设计

许多重的生物过程,如酶原激活、肽激素合成、病毒蛋白加工和受体成熟,均须蛋白质前体加工酶的剪切处理。因此,蛋白质前体加工酶可能是一种新药开发的对象,综合利用同源模建技术和序列的进化踪迹分析手段,研究了蛋白质前体加工酶furin/kexin与水蛭抑制剂(eglinC）突变体的相互作用模式,阐释furin/kexin各个亚类的底物/抑制剂特异性的共性和差异性的序列结构基础。在此基础上,利用界面再设计策略（核心算法为异型自洽系综最优化）进行了furin/kexin抑制剂的理性再设计,分别以模建的水蛭抑制剂-furin,水蛭 素-kex2复合物结构为模板,对水蛭 抑制剂P1,P2和P4位置进行设计,计算结果显示这三个位置均是偏好碱性残基,与已有的实验结论一致,另外针对furin/kexin各亚类在S′端有较多的特异性残基位置这一特点,对抑制剂P′端的残基位置实施改造,设计furin和kex2的特异性更高的抑制剂,对于furin,设计得到的最好突变体是P2′Glu-P3′Asp-P4′Arg;而对于kex2,最好的突变体是P2′Arg-P3′Arg-P4′Glu。结构分析显示furin和kex2与相应的水蛭 抑制剂突变体形成石油同的相互作用模式,这里我们给出了综合利用同源模建技术,序列的进化踪迹分析和理性再设计进行酶-抑制剂相互作用研究及抑制剂改造的方案;同时提供了合理的理论设计方案。为进一步的实验设计提供理性的指导。     

分子伴侣GroE系统能量传递机制的研究

用SwissPDBViewer软件对分子伴侣GroE系统与底物的相互作用进行了模拟 ,结果表明 :GroEL顶端结构域在GroES和靶蛋白结合之后发生了明显的变化 ;GroEL的cis环上有与三磷酸腺苷ATP相结合的位点 ,ATP水解之后形成的ADP与活性中心的残基相结合 ,而这种结合除导致残基Thr30的构型发生了变化之外 ,其它残基的空间位置和构型基本保持不变 ,暗示其它残基在能量传递过程中形成了刚性骨架 ,而与ADP分子磷酸键结合的残基Thr30则是能量传递的力点。     

应用分子生物学方法鉴定6群肺炎链球菌血清型

目的利用分子生物学方法,鉴定26株6群肺炎链球菌的血清型。方法利用6群肺炎链球菌荚膜多糖相关基因设计合成6对特异性引物,PCR扩增26株肺炎链球菌,并对PCR产物进行基因序列的测定及分析。结果26株肺炎链球菌cpsD蛋白229个氨基酸中有7个突变点,第220~222位均没有缺失;其中,19株肺炎链球菌具有与wci Nα蛋白氨基酸序列完全一致的氨基酸组成,第150位均为Ala,第38位均为Asp,其余7株与wciN_α蛋白氨基酸序列没有相似性,但与wciN_β蛋白氨基酸序列相似性超过99%;15株wciP蛋白第195位为Ser,11株wci P蛋白第195位为Asn。综合分析比对后,26株6群肺炎链球菌中,11株属于6A型肺炎链球菌,8株属于6B型肺炎链球菌,4株属于6C型肺炎链球菌,3株属于6D型肺炎链球菌。结论分子生物学方法可用于6群肺炎链球菌血清型的鉴定,为完善6群肺炎链球菌的菌种档案提供了实验依据。     

白色念珠菌羊毛甾醇14α—去甲基化酶三维结构分子？…

基于四种原核细胞色素Ｐ４５０晶体蛋白Ｐ４５０ＢＭ３、Ｐ４５０ｃａｍ、Ｐ４５０ｔｅｒｐ、４５０ｅｒｙＦ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。建模结果用分     

紫茉莉抗病毒蛋白（Mirabilis antiviral protein，MAP）的空间结构和与底物相互作用的模拟研究

首先应用同源构筑方法建立了低序列同源性的紫茉莉抗病毒蛋白（ＭｉｒａｂｉｌｉｓａｎｔｉｖｉｒａｌＰｒｏｔｅｉｎ,ＭＡＰ）的空间结构,然后对此粗结构进行了分子动力学优化。最后通过对ＭＡＰ,ＴＣＳ与其底物类似物的相互作用,了解ＲＩＰ特异糖苷酶活性的机制。结果表明模拟结构是合理的。ＲＩＰ特异糖苷酶活性主要是由于酶的活性口袋与底物的特殊构象相互匹配。     

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。     

Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.     

Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA+ Box VI and VII motifs

Escherichia coli ATP–DnaA initiates chromosomal replication. For preventing extra‐initiations, a complex of ADP–Hda and the DNA‐loaded replicase clamp promotes DnaA‐ATP hydrolysis, yielding inactive ADP–DnaA. However, the Hda–DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg‐153) and DnaA sensor II Arg‐334 within each AAA⁺ domain are crucial for the DnaA‐ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP–Hda with DnaA requires the Hda residues Ser‐152, Phe‐118 and Asn‐122 as well as Hda Arg‐153 and DnaA Arg‐334. Structural analyses suggest intermolecular interactions between Hda Ser‐152 and DnaA Arg‐334 and between Hda Phe‐118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn‐122 and Arg‐153. These interactions likely sustain a specific association of ADP–Hda and DnaA, promoting DnaA‐ATP hydrolysis. Consistently, ATP–DnaA and ADP–DnaA interact with the ADP–Hda‐DNA–clamp complex with similar affinities. Hda Phe‐118 and Asn‐122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA⁺ proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda–DnaA as well as a potentially fundamental mechanism in AAA⁺ protein interactions.     

Homology modeling and substrate binding study of human CYP2C9 enzyme

The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.     

Homology modeling of a novel epoxide hydrolase (EH) from Aspergillus niger SQ-6: structure-activity relationship in expoxides inhibiting EH activity

The 3D structure of a novel epoxide hydrolase from Aspergillus niger SQ-6 (sqEH) was constructed by using homology modeling and molecular dynamics simulations. Based on the 3D model, Asp191, His369 and Glu343 were predicted as catalytic triad. The putative active pocket is a hydrophobic environment and is rich in some important non—polar residues (Pro318, Trp282, Pro319, Pro317 and Phe242). Using three sets of epoxide inhibitors for docking study, the interaction energies of sqEH with each inhibitor are consistent with their inhibitory effects in previous experiments. Moreover, a critical water molecule which closes to the His369 was identified to be an ideal position for the hydrolysis step of the reaction. Two tyrosine residues (Tyr249 and Tyr312) are able to form hydrogen bonds with the epoxide oxygen atom to maintain the initial binding and positioning of the substrate in the active pocket. These docked complex models can well interpret the substrate specificity of sqEH, which could be relevant for the structural—based design of specific epoxide inhibitors. Figure       

Construction of enzyme-substrate complexes between hen egg-white lysozyme and N-acetyl-D-glucosamine hexamer by systematic conformational search and molecular dynamics simulation

We constructed the complexes between HEWL and (GlcNAc)6 oligomer in order to investigate the amino acid residues related to substrate binding in the productive and nonproductive complexes, and the relationship between the distortion of the GlcNAc residue D and the formation of the productive complexes. We obtained 49 HEWL-(GlcNAc)6 complexes by a systematic conformational search and classified the each one to the three binding modes; left side, center, or right side. Furthermore we performed the molecular dynamics simulation against 20 HEWL-(GlcNAc)6 complexes (8: chair model, 12 : half-chair model) selected from the 49 complexes to investigate the interaction between HEWL and (GlcNAc)6. As results, we confirmed that it is necessary for GlcNAc residue D to be half-chaired form to bind toward the right side to form productive complexes. We found newly that eight amino acid residues interact with the (GlcNAc)6 oligomer, as follows, Arg73, Gly102, Asn103 for GlcNAc residue A; Asn103 for GlcNAc residues B and C; Leu56, Ala107, Val109 for GlcNAc residue D; Ala110 for GlcNAc residue E; and Lys33 for GlcNAc residue F. We also indicated that GlcNAc residue F does not interact with Thr47 and rarely interacts with Phe34 and Asn37.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Homology modeling and molecular dynamics studies of a novel C3-like ADP-ribosyltransferase

The novel C3-like ADP-ribosyltransferase is produced by a Staphylococcus aureus strain that especially ADP-ribosylates RhoE/Rnd3 subtype proteins, and its three-dimensional (3D) structure has not known. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of exoenzyme C3 from Clostridium botulinum (1G24). Then the model structure is further refined by energy minimization and molecular dynamics methods. The putative nicotinamide adenine dinucleotide (NAD(+))-binding pocket of exoenzyme C3(Stau) is determined by Binding-Site Search module. The NAD(+)-enzyme complex is developed by molecular dynamics simulation and the key residues involved in the combination of enzyme binding to the ligand-NAD(+) are determined, which is helpful to guide the experimental realization and the new mutant designs as well. Our results indicated that the key binding-site residues of Arg48, Glu180, Ser138, Asn134, Arg85, and Gln179 play an important role in the catalysis of exoenzyme C3(Stau), which is in consistent with experimental observation.     

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.     

The identification of residues that control signal peptidase cleavage fidelity and substrate specificity

Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at -1 (P1) and small or larger aliphatic residues at the -3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used site-directed mutagenesis, mass spectrometric analysis, and in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile-144 residue. Changes of the Ile-144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the -1 residue in a double mutant in which both Ile-86 and Ile-144 were changed to an alanine. Also, alteration of the Ile-144 and Ile-86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage following a -1 Asn residue. This work shows that Ile-144 and Ile-86 contribute to the signal peptidase substrate specificity and that Ile-144 is important for the accuracy of the cleavage reaction.     

The crystal structure of the inhibitor-complexed carboxypeptidase D domain II and the modeling of regulatory carboxypeptidases

The three-dimensional crystal structure of duck carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both carboxypeptidase D domain I and carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus.