TY - JOUR
T1 - High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues
AU - Szabó, András
AU - Héja, Dávid
AU - Szakács, Dávid
AU - Zboray, Katalin
AU - Kékesi, Katalin A.
AU - Radisky, Evette S.
AU - Sahin-Tóth, Miklós
AU - Pál, Gábor
PY - 2011/6/24
Y1 - 2011/6/24
N2 - 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 (KD 20 pM) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD 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 (Lys51, Arg56, and Arg80) 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.
AB - 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 (KD 20 pM) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD 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 (Lys51, Arg56, and Arg80) 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.
UR - http://www.scopus.com/inward/record.url?scp=79959353475&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79959353475&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.235754
DO - 10.1074/jbc.M111.235754
M3 - Article
C2 - 21515688
AN - SCOPUS:79959353475
SN - 0021-9258
VL - 286
SP - 22535
EP - 22545
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 25
ER -