肽类毒素(蜘蛛、蝎、芋螺)的三维结构

多肽类毒素研究是目前毒素研究的一个重点,对多肽类毒素的三维结构的研究是了解其结构与功能关系的重要基础.对蜘蛛、蝎以及芋螺这3类代表性的有毒动物的多肽类毒素在结构研究方面的进展及其三维结构的特点进行了介绍.其中,蜘蛛毒素多肽分子的结构主要发现有ICK模体(Inhibitor Cystine Knot motif)和D DH模体(disulfided-irectedh-airpin)两类,蝎毒素中长链肽类毒素分子和短链肽类毒素分子的结构明显不同,前者以CSα/β结构模体(Cyss-tabilizedα/βfold m otif)为主,后者则以α/β脚手架结构模体(α/βscaffoldm otif)为主.相对于蜘蛛和蝎而言,芋螺肽类毒素分子的三维结构则表现得更为复杂多样.     

二聚体蝎钠通道毒素的1.4(A)分辨率晶体结构:反常非脯氨酸顺式肽键及其内在结构因素

报道了以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构．蛋白质中的肽键是局部双键,不可旋转,因此具有顺式（cis）和反式（trans）两种构型,它们不能通过旋转操作相互转换．非脯氨酸顺式肽键是指形成该肽键的氨基是由脯氨酸以外的氨基酸提供的（Xaa-nonPro）,这类肽键的顺式构型的自由能远比反式高,因此极少出现在天然蛋白质结构中．事实上,在长时间中,多肽链的“反式肽键连接”被视为蛋白质结构的一条基本规则,把顺式肽键视为不可能．随着高分辨率精确蛋白质结构数量的增加,近年来有详细的统计分析揭示,非脯氨酸顺式肽键（Xaa-nPro）在蛋白质结构中出现的几率为0．03％～0．05％,而且大多存在于功能敏感的结构区域,可能具有重要意义．但由于所用的基本结构数据都来自晶体结构,对这种反常肽键是否由结晶环境影响而形成,存在疑问．此前曾在以单体形式存在的蝎神经毒素mono-BmK M1的高分辨率结构中发现其中肽键Pr09-His10是非脯氨酸顺式肽键,并详细分析了其结构．功能意义．以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构表明,它与mono．BmK M1有不同的空间群、不同的分子堆积方式,不同的晶体环境．结构模型被高度精化,Rcryst达到0．109．dimo-BmK M1结构显示,在不对称单位中的两个M1分子在同一位置（残基9．10之间）都清晰地存在顺式肽键．立体化学分析显示,这一肽键的几何参数和局部结构与mono．BmK M1中的（9．10）顺式肽键基本相同．这一结果表明,非脯氨酸顺式肽键9．10的存在与结晶环境无关,是BmK M1分子的固有结构特征．在此基础上,综合分析了与顺式、反式肽键相关的结构元素,发现与残基（8．19）序列模体-KPXNC-（X为任意氨基酸）所决定的特征回折结构可能是分子内在的主要结构因素,其中第8位残基是Lys或Asp对决定肽键是顺式还是反式有关键作用．近来的突变实验及其晶体结构测定已证实,Lys8／Asp8是（9-10）肽键顺式／反式异构的结构开关,它们对该类分子与不同种属钠通道作用的专一选择性具有重要作用．通过BLAST搜索,发现在其他18个蛋白质中也存在相同的序列模体．KPXNC-,推测在这些蛋白质的相应肽键位置也可能存在反常的脯氨酸顺式肽键。     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

尖吻蝮蛇毒酸性磷脂酶A2的新晶型结构

从尖吻蝮蛇毒中分离出一种酸性磷脂酶A2 ,得到了一种新的晶型。用分子置换法测定其晶体结构 ,结构的分辨率达到 0 .2 8nm。该结构给出 2 1.9%的晶体学R因子 (R free =2 5 .7% )和合理的立体化学参数。与已报道的晶型相似 ,不对称单元的两个独立分子形成二体 ,揭示了这种二体的稳定性。二体的形成过程也伴随着 14～ 2 3肽段显著的构象变化 ,这一肽段属于磷脂酶A2 的界面识别部位。氨基酸序列分析表明 ,带正电荷的 34位残基是所有第二类具有溶血活性磷脂酶A2 的共同特点。NaCl对晶体的堆积具有显著的影响 ,从而导致晶胞参数的明显改变。与已报道的晶型不同 ,在酶分子的疏水通道内观测到了 2 甲基 2 ,4戊二醇 (2 methyl 2 ,4 pentanediol,MPD)分子     

The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone

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Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.     

Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae

CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE.     

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQ_δ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.     

Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA

Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 A structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein-DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid-base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5'A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.     

细菌分泌系统的结构及作用机制研究进展

细菌分泌系统（bacterial secretion systems）是一类存在于细菌细胞膜上的大分子复合物，是结构复杂的跨膜分子机器，可为多种细菌的效应物提供分泌途径。目前已经发现了9种细菌分泌系统，即T1SS~T9SS，在细菌的生存及致病力方面发挥着重要作用。随着X射线晶体学（X-ray crystallography）、核磁共振（nuclear magnetic resonance，NMR）及冷冻电镜（cryo-electron microscopy，Cryo-EM）等技术手段的发展与应用，这些大分子复合物的三维结构也得到了一定程度的解析，极大增强了人类对于细菌分泌系统转运底物复杂机制的理解。因此，本文结合近年来关于细菌T1SS~T9SS的研究进展，对各分泌系统的结构信息进行了系统整合，总结了这些分泌系统的分子作用机制，并对其未来的发展方向进行了展望，以期为与细菌蛋白质分泌相关的致病、耐药、环境适应性等机制的研究奠定理论基础，为进一步开发以分泌系统结构为基础的小分子抑菌物质提供精准的三维靶标。     

一个新型耐热普鲁兰酶的结构与功能

新型普鲁兰酶的研究对于普鲁兰酶制剂的国产化、打破国外垄断具有非常重要的意义。从我国云南腾冲地区轮马热泉的淤泥中分离获得了一株耐热普鲁兰酶产生菌LM 18-11,经16S rDNA序列系统进化树分析,确定该菌为厌氧芽胞杆菌Anoxybacillus属种,并从中克隆获得了耐热普鲁兰酶的编码基因,该酶在55℃-60℃、pH 5.6-6.4的范围内具有最大的反应活性。此外,该酶具有较好的热稳定性,在60℃下处理48 h,仍可保持50%以上的活力;动力学分析该酶的Vmax和Km分别为750 U/mg和1.47 mg/mL,是目前文献报道中比活力最高的耐热普鲁兰酶。同时还对该酶进行了晶体结构分析,结果显示该酶具有?-淀粉酶家族中典型的结构,在N端具有一个特殊的底物结合域,该结构域的缺失导致比活力和底物结合力都有相应降低,Vmax和Km分别为324 U/mg和1.95 mg/mL。同时,将该普鲁兰酶编码基因导入枯草芽胞杆菌中,在P43启动子的控制下,普鲁兰酶基因获得了高效表达,胞外酶活可达42 U/mL,相比初始菌种,表达活力提高40倍以上。研究表明该普鲁兰酶具有很好的应用前景。     

抗冻蛋白结构与抗冻机制

抗冻蛋白(amifreeze proteins,AFPs)是20世纪60年代从极地鱼血淋巴中分离的一种大分子抗冻剂,迄今为止科学工作者已从陆地昆虫、植物、细菌和真菌等各类生物中分离到多种抗冻蛋白,并测得了它们的基因序列及一些晶体结构,近些年的工作主要集中在该类蛋白质抗冻机制的研究上。抗冻蛋白具有广泛的应用前景,它不但可以应用于食物的冷鲜贮存及移植器官的低温保存,还可通过转基因提高经济作物的抗冻能力。     

Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases

Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X‐ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology.     

Supramolecular Structure Formed from H-Bonding of Ferrocenyl Carbonyl Propanoic Acid and 4,4'-Bipyridine

Selective recognition in the title compound, (C₁₄H₁₄FeO₃)₂ (C₁₀H₈N₂), between ferrocenyl carbonyl propanoic acid and 4,4'-bipyridine through strong O–-HN intermolecular hydrogen bonds results in a novel supramolecular architecture. Its crystal structure has been solved by single-crystal X-ray diffraction methods while its characterization has been studied by IR and DSC.     

Structure and thermal decomposition of methylamine borane

The crystal structure of methylamine borane has been determined and contains parallel chains of dihydrogen-bonded CH₃NH₂BH₃ molecules. Thermal decomposition takes place from the melt (ΔH_fusion = 8.5 kJ mol⁻¹) and begins with the formation of an ionic borohydride. Hydrogen is liberated in two stages, at ca. 100 and 190 °C, with the observed rates during the first stage (ΔH = −25 kJ mol⁻¹, E_a = 115 kJ mol⁻¹) strongly dependent on temperature and time. cis- and trans-N-trimethylcyclotriborazane are formed during the first stage and subsequently cross-link to yield a non-volatile solid. Before this cross-linking, the system exhibited a high degree of volatility, with weight losses in excess of 80% observed in TG experiments using flowing gas.     

Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (∼70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of the P1 residue for protease inhibition. This study provides important information for the development of peptidic agents for pharmacological intervention.     

Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.     

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Bacterial l-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig l-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by l-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for l-asparaginases.     

Crystal structure of the autocatalytic initiator of glycogen biosynthesis,glycogenin

The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.     

Insights into the fatty acid chain length specificity of the carboxylesterase PA3859 from Pseudomonas aeruginosa: A combined structural,biochemical and computational study

The open reading frame PA3859 of Pseudomonas aeruginosa encodes an intracellular carboxylesterase belonging to a group of microbial enzymes (EC 3.1.1.1) that catalyze the hydrolysis of aliphatic and aromatic esters with a broad substrate specificity. With few exceptions, for this class of enzymes, belonging to the α/β-hydrolase fold superfamily, very little information is available regarding their biochemical activity and in vivo function. The X-ray crystal structure of recombinant PA3859 has been determined for two crystal forms (space groups P2₁ and P2₁2₁2). The kinetic properties of the enzyme were studied using p-nitrophenyl esters as substrates and data fitted to a surface dilution mixed micelle kinetic model. Enzymatic assays and computational docking simulations, pinpointed the enzyme’s preference for esters of palmitic and/or stearic acids and provided insights into the enzyme–substrate favorable binding modes.