title: High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4' acidic residues.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
authors.forename: András
authors.initials: A
authors.lastname: Szabó
authors.forename: Dávid
authors.initials: D
authors.lastname: Héja
authors.forename: Dávid
authors.initials: D
authors.lastname: Szakács
authors.forename: Katalin
authors.initials: K
authors.lastname: Zboray
authors.forename: Katalin A
authors.initials: KA
authors.lastname: Kékesi
authors.forename: Evette S
authors.initials: ES
authors.lastname: Radisky
authors.forename: Miklós
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: Gábor
authors.initials: G
authors.lastname: Pál
mesh_terms: D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D002918:Chymotrypsin; D019476:Insect Proteins; D019151:Peptide Library; D010455:Peptides; D011480:Protease Inhibitors; C117569:chymotrypsin C; C113210:LOC126284306 protein, Schistocerca gregaria