IMPORTANCE OF THE EVOLUTIONARILY CONSERVED GLYCINE RESIDUE IN THE N-TERMINAL REGION OF HUMAN CYSTATIN-C (GLY-11) FOR CYSTEINE ENDOPEPTIDASE INHIBITION
IMPORTANCE OF THE EVOLUTIONARILY CONSERVED GLYCINE RESIDUE IN THE N-TERMINAL REGION OF HUMAN CYSTATIN-C (GLY-11) FOR CYSTEINE ENDOPEPTIDASE INHIBITION
复制标题
DOI：
10.1042/bj2910123
复制
发表时间：
1993-04-01
影响因子：
4.1
通讯作者：
ABRAHAMSON, M
中科院分区：
文献类型：
--
作者：
HALL, A;DALBOGE, H;ABRAHAMSON, M
人胱抑素C变体，其中进化上保守的Gly-11残基已经被具有带正电荷（Arg）、带负电荷（Glu）、大疏水性（Trp）或小（Ser或Ala）侧链的残基取代，已经通过定点诱变产生并在大肠杆菌中表达。分离并结构验证了这五种变体。通过测定它们与半胱氨酸内肽酶木瓜蛋白酶和人组织蛋白酶B以及与半胱氨酸外肽酶二肽基肽酶I的复合物的解离平衡常数（K（i）），将它们的抑制特性与野生型重组半胱氨酸蛋白酶抑制剂C的抑制特性进行比较。Ser-11和Ala-11半胱氨酸蛋白酶抑制剂C变体显示两种内肽酶的K（i）值约为1.00。20-比野生型半胱氨酸蛋白酶抑制剂C的那些高1倍，而Trp-11、Arg-11和Glu-11变体的相应值增加了约2000倍。相反，所有五种变体与外肽酶相互作用的K（i）值与野生型胱抑素C的K（i）值相差不到10倍。将野生型胱抑素C和Ser-11、Ala-11和Glu-11变体与中性粒细胞弹性蛋白酶一起孵育，这在所有情况下导致氨基酸残基10和11之间的单个肽键的快速水解。这三种N-末端十肽缺失的半胱氨酸蛋白酶抑制剂C变体与木瓜蛋白酶相互作用的K（i）值为20-50 nM，仅比N-末端截短的野生型半胱氨酸蛋白酶抑制剂C的值高一个数量级，而野生型半胱氨酸蛋白酶抑制剂C的值又与全长Glu-11、Arg-11和Trp-11变体的相应值相似。这些数据表明，野生型胱抑素C的位置11中的保守Gly残基的关键特征是，该残基缺乏侧链，将允许胱抑素C的N-末端区段采用适合于与半胱氨酸内肽酶的底物结合口袋相互作用的构象，导致高亲和力结合和有效抑制。蛋白酶接触区的剩余部分的功能特性，这是建立从更多的C-末端抑制剂片段，没有显着影响，即使当氨基酸与庞大的或带电的侧链取代甘氨酸-11残基的N-末端片段。
Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (K(i)) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed K(i) values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11, Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the K(i) values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The K(i) values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.