Eglin C与2709碱性蛋白酶相互作用分析及亲和纯化方法

Eglin C是来自水蛭中的一种小型热稳定蛋白质,属于马铃薯胰凝乳蛋白酶抑制剂家族,可以抑制弹性蛋白酶、枯草杆菌蛋白酶、组织蛋白酶、α-lytic蛋白酶以及胰凝乳蛋白酶等。然而,利用eglin C纯化蛋白酶,尚未见研究报道。本文将化学合成的编码 eglin C及其突变体的基因序列,克隆到原核表达载体pQE30,在大肠杆菌BL21获得His6-Tag-eglin C及其突变体的重组蛋白质。SDS-PAGE显示,eglin C蛋白的分子量大约8 kD。His6-Tag-eglin C对胰凝乳蛋白酶、地衣芽孢杆菌2709碱性蛋白酶、枯草芽孢杆菌PB92碱性蛋白酶、枯草杆菌中性蛋白酶的半抑制剂浓度（IC50）分别为0.20±0.15、0.24±0.19、3.33±0.47和52.46±0.38 μmol/L。利用分子对接、基因突变以及荧光光谱等,分析eglin C及其突变体与2709蛋白酶的相互作用。结果显示,2709碱性蛋白酶对eglin C荧光淬灭属于静态淬灭,解离常数为2.60×10-7 mol/L,eglin C中的Asn50 残基对eglin C和2709碱性蛋白酶的结合发挥重要作用。利用eglin C与蛋白酶的特异结合的特性,构建亲和纯化载体,用于纯化来源于地衣芽孢杆菌的2709碱性蛋白酶,相比常规的蛋白酶纯化,显著简化了操作步骤。     

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。     

Kunitz 型丝氨酸蛋白酶抑制剂结构与功能研究

蛋白酶抑制剂在酶学及蛋白质的结构与功能关系研究中有重要意义,Kunitz型丝氨酸蛋白酶抑制剂是其中最重要的,也是研究最广泛的蛋白酶抑制剂之一．该类蛋白酶抑制剂三维结构高度保守：由一个明显的疏水核心、三对高度保守的二硫键桥、三链β-折叠和一个N端3 10螺旋及一个C端α-螺旋组成．3对二硫键对分子空间结构的稳定起着非常重要的作用．这一类型抑制剂有5个主要的活性位点：P1、P1’、P3、P3’、P4,它们都位于一个溶剂暴露的环上．P1位点是抑制作用的关键活性位点,抑制剂的专一性由P1位点氨基酸残基的性质决定;P1’位点氨基酸残基的侧链大小对抑制剂．酶的结合常数有很大影响,用大的侧链残基取代会导致结合常数降低;P4位点残基被取代经常产生负效应,会导致活性区域环的构象发生很大改变,从而影响酶与抑制剂的结合．     

大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析

为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH, d-3-phosphoglycerate dehydrogenase, EC 1.1.1.95）,通过对其碱基序列和蛋白质结构分析,用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407~410位氨基酸）、M3（缺失337~410位氨基酸）。M0(野生型)及各突变型基因与pET22b(+)载体连接后,表达融合蛋白。在非变性条件下,由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白。酶活性测定结果表明,M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性,且部分解除了终产物L-丝氨酸的反馈抑制作用;M3蛋白酶完全解除了终产物的反馈抑制作用,但酶本身的催化活性略有降低（为野生型的83%）。M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7 μmol/L、20 μmol/L、50 μmol/L,说明该酶C-末端1~4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响。     

枯草杆菌蛋白酶的基因工程改性

枯草杆菌蛋白酶（Ｓｕｂｔｉｌｉｓｉｎ）是一种工业上应用很广的酶,在洗涤剂、制革、丝绸等多种行业上有着广泛的用途。特别是用于生产加酶洗涤剂,帮助去除血渍、奶渍、汗渍及可可等各种蛋白污垢。１９６０年,丹麦人首先利用地衣芽孢杆菌生产了被称为Ｓｕｂｔｉｌｉｓｉｎ Ｃａｒｌｓｂｅｒｇ的碱性蛋白酶,随后该酶被用于生产加酶洗涤剂,目前有资料称国外市场上９０％是加酶洗涤剂。国内１９９０年枯草杆菌蛋白酶产量约为１．３万吨,加酶洗衣粉占洗涤剂总量约１０％。枯草杆菌蛋白酶的生产菌和研究对象主要是地衣芽孢杆菌、解淀粉芽…     

一新中温碱性蛋白酶基因的克隆及原核表达

摘要:【目的】从环境中分离筛选产蛋白酶、降解蛋白质的菌株,寻找使用价值较高的碱性蛋白酶。【方法】通过酪蛋白平板法分离筛选产蛋白酶菌株,经生理生化方法及16S rDNA 基因序列鉴定菌株;利用简并引物及基因组步移克隆蛋白酶完整开放阅读框;蛋白酶前体蛋白及成熟肽序列在大肠杆菌(Escherichia coli) BL21(DE3)中进行重组表达;纯化活性蛋白酶后,利用化学合成多肽底物(succinyl-Ala-Ala-Pro-Phe-p-nitroanilide)检测酶活性质及其催化活力。【结果】分离到的菌株L010被鉴定命名为芽胞杆菌( Bacillus sp.)L010;蛋白酶开放阅读框包含了1149个碱基,编码382个氨基酸,氨基酸序列按其功能分为N端的30个氨基酸残基组成的信号肽,77个氨基酸残基构成的前导肽,C端275个氨基酸残基组成的成熟肽;此蛋白属于丝氨酸蛋白酶家族中枯草杆菌蛋白酶类(Subtilisins)成员,并命名为SprD;SprD的前体蛋白在大肠杆菌(Escherichia coli)BL21(DE3)中重组表达时,在前导肽辅助下自加工为活性蛋白酶;SprD呈现出较高的催化活力,其反应最适条件为温度70℃,pH9－10。【结论】SprD在碱性(pH 7.0－ 10.0)、中高温(25℃－60℃)条件下的稳定性及较高的催化能力使其具有一定的研究和潜在利用价值。     

枯草杆菌碱性蛋白酶Ki 2基因的序列测定及M222A定点突变

含有枯草杆菌碱性蛋白酶Ｋｉ-２基因的１.９ｋｂＤＮＡ片段用限制酶切成几个小片段,将这些片段分别插入Ｍ１３ｍｐ１８或Ｍ１３ｍｐ１９中,用通用测序引物测得全序列。所得全序列与蛋白酶Ｅ相比较,在结构基因部分仅有８个碱基不同,由此而导致两个氨基酸的差异。此１.９ｋｂ的片段插入枯草杆菌大肠杆菌穿梭质粒Ｐｂｅ-２,得到的重组质粒转化蛋白酶缺陷型的枯草芽孢杆菌ＤＢ１０４,结果表明枯草杆菌碱性蛋白酶Ｋｉ-２基因在ＤＢ１０４中能利用自身的调控元件表达并分泌到胞外。将Ｋｉ-２蛋白酶的２２２位甲硫氨酸突变成丙氨酸,突变后的Ｋｉ-２蛋白酶具有抗氧化性,但比活性比野生型的约低１倍     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

Potato I型蛋白酶抑制剂 rBTI及其突变体的表达、纯化和活性研究

荞麦胰蛋白酶抑制剂(BTI)属于丝氨酸蛋白酶抑制剂Potato I家族,典型构象中有1段暴露在分子外侧的结合区,该区内P1′、P2、P6′与P8′位的氨基酸残基具有高度保守性.本文依据近期解析的rBTI晶体结构以及rBTI与胰蛋白酶复合物晶体结构信息,对rBTI 中的P2和P8′位氨基酸进行突变,构建了pExSecI- Bti-P44T 和 pExSecI-Bti-W53R 重组质粒,转入大肠杆菌 BL21（DE3）中进行表达,通过Resource Q阴离子交换层析和Superdex G 75 HR 10/300凝胶柱进行分离纯化后,测定了rBTI及其突变体对胰蛋白酶的抑制常数,以及它们对HepG2 细胞内的蛋白酶体和细胞增殖的抑制作用. 实验结果显示,rBTI 的44和53位分别突变为 Thr和Arg后,Ki 分别为2.91×10-9mol/L和2.97 ×10-7mol /L,前者较rBTI（Ki = 3.56×10-8 mol/L）降低1个数量级,而后者较rBTI升高1个数量 级.功能分析显示,rBTI及2种突变体对HepG2细胞内的蛋白酶体基本没有抑制作 用,但是它们都保留了对HepG2细胞增殖的抑制活性.这些结果揭示,作为一种特异的胰蛋白酶抑制剂,rBTI分子中的保守区域氨基酸残基虽然对胰蛋白酶的抑制作用有显著影响,但并不影响其抑制肿瘤细胞的增殖,仍能发挥其原有的生物学功能.     

枯草杆菌蛋白酶突变体的纯化和晶体生长

应用基因工程手段,获得了枯草杆菌蛋白酶Ｅ的双突体基因（Ｍ２２２Ａ,Ｎ１１８Ｓ）,此基因在枯草芽孢杆菌表达得到了既抗氧又耐高温的碱性蛋白酶。含Ｍ２２２,Ｎ１１８Ｓ碱性蛋白酶基因的枯草杆菌发酵液经过硫酸铵分级沉淀和ＤＥＡＥＳｅｐｈａｄｅｘＡ－２５阴离子交换层析柱,再在ＦＰＬＣ层析系统直用Ｈｉｌｏａｄ２６／１０ＳＳｅｐｈａｒｏｓｅＨＰ阳离了交换柱分离得到ＳＤＳ－ＰＡＧＥ电泳纯的蛋白酶样品,突变体酶的等电     

Two engineered eglin c mutants potently and selectively inhibiting kexin or furin

Eglin c with mutants L45R and D42R at the P(1) and P(4) positions has been reported to become a stable inhibitor toward the proprotein convertases (PC), furin and kexin, with a K(i) of 2.3x10(-8) and 1.3x10(-10) M, respectively. The mutant was further engineered at the P(2)'-P(4)' positions to create a more potent and selective inhibitor for each enzyme. The residue Asp at P(1)' which is crucial for stabilizing the conformation of eglin c remained unchanged. The eglin c mutants cloned into the vector pGEX-2T and expressed in Escherichia coli (DH5alpha) were purified to homogeneity, and their inhibitory activities toward the purified recombinant furin and kexin were examined. The results showed that (1) Leu47 at P(2)' replaced with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes; (2) the replacement of Arg with Asp at P(3)' was favorable for inhibiting furin with a K(i) of 7.8 x 10(-9) M, but not for inhibiting kexin; (3) the replacement of Tyr with Glu at P(4)' increased the inhibitory activity to kexin with a K(i) of 3 x 10(-11) M, but was almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site-directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P' positions.     

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.     

Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin

We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases. A structural gene was synthesized that encoded "R(1)-eglin", having Arg at P(1) in the reactive site loop in place of Leu(45). Ten additional variants were created by cassette mutagenesis of R(1)-eglin. These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined. R(1)-eglin itself was a modest inhibitor of Kex2, with a K(a) of approximately 10(7) M(-)(1). Substituting Arg (in R(4)R(1)-eglin) or Met (in M(4)R(1)-eglin) for Pro(42) at P(4) created potent Kex2 inhibitors exhibiting K(a) values of approximately 10(9) M(-)(1). R(4)R(1)-eglin inhibited furin with a K(a) of 4.0 x 10(8) M(-)(1). Introduction of Lys at P(1), in place of Arg in R(4)R(1)-eglin reduced affinity only approximately 3-fold for Kex2 but 15-fold for furin. The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis. R(4)R(1)-eglin formed stable 1:1 complexes with both Kex2 and furin. However, substitution of Lys at P(2) in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition). Surprisingly, R(6)R(4)R(1)-eglin, in which Arg was substituted for Gly(40) in R(4)R(1)-eglin, exhibited stable, high-affinity complex formation with Kex2 (K(a) of 3.5 x 10(9) M(-)(1)) but temporary inhibition of furin. This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate. Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases.     

Quantitative characterization of furin specificity. Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides.

Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis. We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates. In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates. With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-1). Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition. Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue. We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4). The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.     

Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway.

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.     

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.     

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics refinement of a thermitase-eglin-c complex at 1.98 A resolution and comparison of two crystal forms that differ in calcium content

The crystal structure of the complex of thermitase with eglin-c in crystal form II, obtained in the presence of 5 mM-CaCl2, has been determined at 1.98 A resolution. The structure was solved by a molecular replacement method, then molecular dynamics crystallographic refinement was started using the thermitase-eglin-c structure as determined for crystal form I. A ten degrees rigid body misplacement of the core of eglin-c was corrected by the molecular dynamics crystallographic refinement without any need for manual rebuilding on a graphics system. A final crystallographic R-factor of 16.5% was obtained for crystal form II. The comparison of the complexes of thermitase with eglin-c in the two crystal forms shows that the eglin-c cores are differently oriented with respect to the protease. The inhibiting loop of eglin binds in a similar way to thermitase as to subtilisin Carlsberg. A tryptophanyl residue at the S4 site explains the preference of thermitase for aromatic residues of the substrate at the P4 site. The difference in the P1 binding pocket, asparagine in thermitase instead of glycine in subtilisin Carlsberg, does not change the binding of eglin-c. The preference for an arginyl residue at the P1 site of thermitase can be explained by the hydrogen bonding with Asn170 in thermitase. Three ion-binding sites of thermitase have been identified. The strong and weak calcium-binding sites resemble the equivalent sites of subtilisin Carlsberg and subtilisin BPN', though there are important amino acid differences at the calcium-binding sites. The medium-strength calcium-binding site of thermitase is observed in the subtilisin family for the first time. The calcium is bound to residues from the loop 57 to 66. A difference in chelation is observed at this site between the two crystal forms of thermitase, which differ in calcium concentration. Additional electron density is observed near Asp60 in crystal form II, which has more calcium bound than form I. This density is possibly due to a water molecule ligating the calcium ion or the result of Asp60 assuming two significantly different conformations.     

Effects of eglin-c binding to thermitase: three-dimensional structure comparison of native thermitase and thermitase eglin-c complexes.

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin-c, grown from three different calcium concentrations. It appeared that the B-factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably similar to those of the 1.4 A native structure: the main chain atoms have an rms difference of only 2.3 A2; for all atoms this difference is 4.6 A2. The rms positional differences between these two structures of thermitase are 0.31 A for the main chain atoms and 0.58 A for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X-ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect. A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rms deviation of 0.13 A for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 A in C alpha positions. These are probably due to crystal packing effects. The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.