High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues
High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues
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DOI：
10.1074/jbc.m111.235754
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发表时间：
2011-06-24
影响因子：
4.8
通讯作者：
Pal, Gabor
中科院分区：
文献类型：
--
作者：
Szabo, Andras;Heja, David;Pal, Gabor
人胰凝乳蛋白酶C （chymotrypsin C， CTRC）是一种参与调节肠道消化酶活性的胰腺蛋白酶。其他凝乳胰酶和弹性酶在CTRC切割的调控位点上无活性，表明CTRC识别独特的序列模式。为了表征CTRC特异性的分子决定因素，我们从显示SGPI-2变体的噬菌体文库中选择了针对CTRC的高亲和力底物样小蛋白抑制剂，SGPI-2是一种来自grestocerca gregaria的天然胰凝血酶抑制剂。根据所选择的序列模式，我们设计了8种抑制剂变体，其中P1 (Met或Leu), P2‘ （Leu或Asp）和P4’ （Glu， Asp或Ala）的活性环中的氨基酸残基不同。与CTRC的结合实验表明(1)含有P1上Leu的抑制剂的结合能力比含有P1 Met的抑制剂强10倍；（ii） P2‘位点的Asp（相对于Leu）降低了亲和力，但增加了选择性；（iii） Glu或P4’位点的Asp（相对于Ala）使亲和力提高了10倍。最高亲和力的SGPI-2变体（K-D 20 pM）与CTRC的结合比亲本分子紧密575倍。最具选择性的抑制剂变体对其他人类凝乳胰蛋白酶和弹性酶的K-D值为110 pM，选择性范围为225- 112,664倍。同源性建模和诱变在人类CTRC表面发现了一簇碱性氨基酸残基(Lys(51), Arg（56）和Arg(80))，它们与抑制剂的P4'酸性残基相互作用。CTRC在P4'的酸性偏好在胰腺蛋白酶中是独特的，这可能有助于CTRC介导的消化酶调节的高特异性。
Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K-D 20 pM) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K-D of 110 pM and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.