Active site dynamics of acyl-chymotrypsin

The motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl-chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH^? for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His-57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the N^ε2 of His-57 and is heldin place through a network of hydrogen-bonded water molecules in theactive site. The water molecule was found as close as 2.8 Å to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His-57-N^ε2 and Ser-195-0^γ with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His-57 imidazole ring between positions suitable for providing general-base catalyzed assistance and for providing general-acid catalyzed assistance may notbe required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His-57, Ser-195, and Asp-102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X-ray crystal-lographic data of γ-chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site. © Wiley-Liss, Inc.     

蛋白酶及其抑制剂关键活性位点研究进展

蛋白酶广泛存在于生物体中,参与分解蛋白质,维持生物体正常的生命活动.蛋白酶抑制剂通过与蛋白酶活性位点结合调控靶蛋白酶活性,从而影响蛋白质代谢.蛋白酶及其抑制剂关键氨基酸的突变,可以影响其生理功能、稳定性、催化活性、抑制特异性等.通过挖掘自然界蛋白酶及其抑制剂的各种突变体,分析它们的关键活性位点,并运用蛋白质工程手段改造...     

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．     

野生型和突变型荞麦蛋白酶抑制剂的活性比较及抗肿瘤功能分析

运用定点突变技术研究重组荞麦胰蛋白酶抑制剂(rBTI)的作用位点,先后构建了R45A-aBTI 和R45F-fBTI 两个突变体．抑制活性测定显示,aBTI 和fBTI 均丧失了胰蛋白酶抑制活性,却分别增加了对弹性蛋白酶和胰凝乳蛋白酶的抑制活性,确定Arg⁴⁵为野生型rBTI 的作用位点．稳定性分析表明,rBTI、aBTI 和fBTI 均具有很高的热稳定性及酸碱稳定性．采用MTT 比色法分别检测野生型和突变型抑制剂对肿瘤细胞生长的抑制作用．结果表明,突变前后的3种抑制剂对HL-60 和EC9706 细胞的生长均显示出很强的抑制作用,并且具有明显的浓度依赖性和时间依赖性．通过研究不仅确定了rBTI 的作用位点,而且获得了两种新型蛋白酶抑制剂,且作用位点的改变并不影响其对肿瘤细胞的生长抑制作用．为进一步研究胰蛋白酶抑制剂的结构与功能的关系以及抗肿瘤药物的研制提供了新的思路．     

Single point mutation induced alterations in the equilibrium structural transitions on the folding landscape of HIV-1 protease

Equilibrium folding–unfolding transitions are hard to study in HIV-1 protease (PR) because of its autolytic properties. Further, the protease exhibits many tolerant point mutations some of which also impart drug resistance to the protein. It is conceivable that the mutations affect protein's function by altering its folding characteristics; these would clearly depend on the nature of the mutations themselves. In this background, we report here NMR studies on the effects of D25?N mutation, which removes one negative charge from the protein at the active site, on the equilibrium folding behaviour of PR starting from its acetic acid denatured state. It is observed that in PR_D25N two slowly exchanging conformations are present at the N-terminal. One of them is similar to that of PR. Though the conformational and dynamics preferences of PR and PR_D25N are fairly similar in 9?M acetic acid, they seem to undergo different folding transitions when acetic acid concentration is reduced. The differences are seen in the active site, in the flap, and in the hinge of the flap regions. The present study suggests that such differences, though different in detail, would occur for other mutations as well, and also for different initial denatured states. These would have significant regulatory implications for the efficacy of protease function.     

1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin

Under the selection pressure of drugs, mutations appear in HIV-1 protease even at the sites, which are conserved in the untreated individuals. Cysteine 95 is a highly conserved residue and is believed to be involved in regulation of HIV-1 protease. In some of the virus isolates from patients undergoing heavy treatment with anti-HIV protease drugs, C95F mutation has appeared. The present study reports 1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin. It is found that in this mutant, dimer interface has become more rigid and that the packing at the interface of terminal and core domains is altered. These alterations may be relevant to C95F mutation conferring drug resistance to HIV-1 protease.     

Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold

Summary The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV-cell interactions and as a lead in the development of antiviral drugs.     

Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold

The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV–cell interactions and as a lead in the development of antiviral drugs.     

Inhibition of catalytic activities of botulinum neurotoxin light chains of serotypes A,B and E by acetate,sulfate and calcium

The catalytic domain, known as light chain (Lc), of the most poisonous botulinum neurotoxins (BoNTs), possesses endoprotease activity that triggers the ultimate poisonous effect to animals and humans. X-ray crystallographic structure of Lc of several BoNT serotypes has identified at least four small ligands at or near the respective active sites. They are sulfate ions in LcA, LcB, and LcE; an acetate ion in LcA; a calcium ion in LcB; and a potassium ion in LcD. Roles of these ligands on the structure and function of the proteins are not known. We have investigated the roles of sulfate, acetate, and calcium on the catalytic activities of LcA, LcB, and LcE using 17-35-residue synthetic peptide substrates. All three ligands inhibited all Lc activities. For LcA and LcB, the order of inhibition effectiveness was calcium>sulfate>acetate. The inhibition effectiveness expressed as IC₅₀, did not correlate with the occurrence or proximity of the ions to the active site. Moreover, addition of acetate or sulfate to LcA did not affect the near-UV circular dichroism spectra, tryptophan, and tyrosine fluorescence spectra, and mid points of thermal denaturation of LcA. Our results suggest that acetate, sulfate, and calcium nonspecifically interact with BoNT Lc, and their occurrence in the crystal structures could have been due to opportunistic binding to complementary pockets.     

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

重组旋毛虫plancitoxin-1-like活性位点突变体蛋白的核酸酶活性

旋毛虫plancitoxin-1-like(Ts-Pt)是旋毛虫125种DNaseⅡ家族蛋白中唯一具有典型DNaseⅡ活性区域HKD基序的核酸酶,且普遍认为,组氨酸位点是DNaseⅡ的活性氨基酸位点。为研究Ts-Pt活性位点突变体蛋白的核酸酶活性,利用重叠PCR方法获得Ts-Pt活性位点突变体片段,以p ET-28a(+)为载体构建重组表达质粒并在大肠杆菌中诱导表达。重组Ts-Pt突变体蛋白经亲和层析纯化后进行SDS-PAGE分析。利用琼脂糖凝胶电泳法和核酸酶酶谱分析重组Ts-Pt突变体蛋白的核酸酶活性。成功构建含Ts-Pt突变体重组质粒的基因工程菌,SDS-PAGE和亲和层析纯化结果显示,重组Ts-Pt突变体蛋白呈包涵体表达。重组蛋白经复性后并没有表现出核酸酶活性,但核酸酶酶谱分析结果显示,包涵体表达的重组Ts-Pt突变体蛋白表现出降解DNA的能力。同时,N端和C端活性位点H及HCK和DHSK突变并不影响Ts-Pt的核酸酶活性,研究结果为进一步研究庞大的DNaseⅡ家族蛋白在旋毛虫发育和感染方面的作用提供一定的参考。     

Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

The increasing emergence of drug-resistant tuberculosis （TB） poses a serious threat to the control of this disease. It is in urgent need to develop new TB drugs. Tryptophan biosynthetic pathway plays an important role in the growth and replication of Mycobacterium tuberculosis （Mtb）. The β-subunit of tryptophan synthase （TrpB） catalyzes the last step of the tryptophan biosynthetic pathway, and it might be a potential target for TB drug design. In this study, we overexpressed, purified, and characterized the putative TrpB-encoding gene Rv1612 in Mtb H37Rv. Results showed that Mtb His-TrpB optimal enzymatic activity is at pH 7.8 with 0.15 M Na^＋ or 0.18 M Mg^2＋ at 37℃. Structure analysis indicated that Mtb TrpB exhibited a typical β/α barrel structure. The amino acid residues believed to interact with the enzyme cofactor pyridoxal-5＇-phosphate were predicted by homology modeling and structure alignment. The role of these residues in catalytic activity of the Mtb His-TrpB was confirmed by site-directed mutagenesis. These results provided reassuring structural information for drug design based on TrpB.