Hall A, Dalbøge H, Grubb A, Abrahamson M
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):123-9. doi: 10.1042/bj2910123.
Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by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 (Ki) 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 Ki 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 Ki 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 Ki 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.
通过定点诱变并在大肠杆菌中表达，已产生了人胱抑素C变体，其中进化上保守的甘氨酸-11残基已被带正电荷（精氨酸）、带负电荷（谷氨酸）、大体积疏水（色氨酸）或小（丝氨酸或丙氨酸）侧链的残基取代。分离出这五个变体并进行了结构验证。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以及半胱氨酸外肽酶二肽基肽酶I形成的复合物的解离平衡常数（Ki），将它们的抑制特性与野生型重组胱抑素C的抑制特性进行了比较。丝氨酸-11和丙氨酸-11胱抑素C变体对这两种内肽酶的Ki值比野生型胱抑素C的Ki值高约20倍，而色氨酸-11、精氨酸-11和谷氨酸-11变体的相应值增加了约2000倍。相反，所有五个变体与外肽酶相互作用的Ki值与野生型胱抑素C的Ki值相差不到10倍。将野生型胱抑素C以及丝氨酸-11、丙氨酸-11和谷氨酸-11变体与中性粒细胞弹性蛋白酶一起孵育，在所有情况下均导致第10和11位氨基酸残基之间的单个肽键快速水解。这三种缺乏N端十肽的胱抑素C变体与木瓜蛋白酶相互作用的Ki值为20 - 50 nM，仅比N端截短的野生型胱抑素C的值高一个数量级，而后者又与全长谷氨酸-11、精氨酸-11和色氨酸-11变体的相应值相似。这些数据表明，野生型胱抑素C第11位保守甘氨酸残基的关键特征是该残基没有侧链，这将使胱抑素C的N端片段能够采取适合与半胱氨酸内肽酶的底物结合口袋相互作用的构象，从而实现高亲和力结合和有效抑制。即使带有大体积或带电荷侧链的氨基酸取代了N端片段的甘氨酸-11残基，由更多C端抑制剂片段构成的蛋白酶接触区域其余部分的功能特性也不会受到显著影响。
