High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues

András Szabó, Dávid Héja, Dávid Szakács, Katalin Zboray, K. Kékesi, Evette S. Radisky, Miklós Sahin-Tóth, G. Pál

Research output: Contribution to journalArticle

21 Citations (Scopus)

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 (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.

Original languageEnglish
Pages (from-to)22535-22545
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number25
DOIs
Publication statusPublished - Jun 24 2011

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Bacteriophages
Display devices
Proteins
Viperidae
Pancreatic Elastase
Chymotrypsin
Peptide Hydrolases
chymotrypsin C
Basic Amino Acids
Mutagenesis
Enzyme activity
Enzymes
Amino Acids
Molecules

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues. / Szabó, András; Héja, Dávid; Szakács, Dávid; Zboray, Katalin; Kékesi, K.; Radisky, Evette S.; Sahin-Tóth, Miklós; Pál, G.

In: Journal of Biological Chemistry, Vol. 286, No. 25, 24.06.2011, p. 22535-22545.

Research output: Contribution to journalArticle

Szabó, András ; Héja, Dávid ; Szakács, Dávid ; Zboray, Katalin ; Kékesi, K. ; Radisky, Evette S. ; Sahin-Tóth, Miklós ; Pál, G. / High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 25. pp. 22535-22545.
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