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Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors.

Salameh MA, Soares AS, Alloy A, Radisky ES
Protein Sci 21 1103-12 (2012)
Cited: 17 times
EuropePMC logo PMID: 22610453

Abstract

Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P(4) ' residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.

Reviews - 4dg4 mentioned but not cited (1)

  1. Biochemical and structural insights into mesotrypsin: an unusual human trypsin. Salameh MA, Radisky ES. Int J Biochem Mol Biol 4 129-139 (2013)

Articles - 4dg4 mentioned but not cited (2)

  1. Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors. Salameh MA, Soares AS, Alloy A, Radisky ES. Protein Sci. 21 1103-1112 (2012)
  2. Rapid in silico Design of Potential Cyclic Peptide Binders Targeting Protein-Protein Interfaces. Santini BL, Zacharias M. Front Chem 8 573259 (2020)


Articles citing this publication (14)

  1. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy. Cohen I, Kayode O, Hockla A, Sankaran B, Radisky DC, Radisky ES, Papo N. Biochem. J. 473 1329-1341 (2016)
  2. Sequence and conformational specificity in substrate recognition: several human Kunitz protease inhibitor domains are specific substrates of mesotrypsin. Pendlebury D, Wang R, Henin RD, Hockla A, Soares AS, Madden BJ, Kazanov MD, Radisky ES. J. Biol. Chem. 289 32783-32797 (2014)
  3. Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates. Alloy AP, Kayode O, Wang R, Hockla A, Soares AS, Radisky ES. J. Biol. Chem. 290 21523-21535 (2015)
  4. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis. Kayode O, Wang R, Pendlebury DF, Cohen I, Henin RD, Hockla A, Soares AS, Papo N, Caulfield TR, Radisky ES. J. Biol. Chem. 291 26304-26319 (2016)
  5. Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis. Szabó A, Ludwig M, Hegyi E, Szépeová R, Witt H, Sahin-Tóth M. J. Biol. Chem. 290 17282-17292 (2015)
  6. Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin. Cohen I, Coban M, Shahar A, Sankaran B, Hockla A, Lacham S, Caulfield TR, Radisky ES, Papo N. J Biol Chem 294 5105-5120 (2019)
  7. Purification and characterization of cocoonase from the silkworm Bombyx mori. Fukumori H, Teshiba S, Shigeoka Y, Yamamoto K, Banno Y, Aso Y. Biosci. Biotechnol. Biochem. 78 202-211 (2014)
  8. Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases. Cohen I, Naftaly S, Ben-Zeev E, Hockla A, Radisky ES, Papo N. Biochem. J. 475 1335-1352 (2018)
  9. Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site. Lacham-Hartman S, Shmidov Y, Radisky ES, Bitton R, Lukatsky DB, Papo N. PLoS One 16 e0249616 (2021)
  10. Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes. Heyne M, Shirian J, Cohen I, Peleg Y, Radisky ES, Papo N, Shifman JM. J Am Chem Soc 143 17261-17275 (2021)
  11. Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens. Moreau T, Recoules E, De Pauw M, Labas V, Réhault-Godbert S. Poult Sci 103 103182 (2023)
  12. Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries. Naftaly S, Cohen I, Shahar A, Hockla A, Radisky ES, Papo N. Nat Commun 9 3935 (2018)
  13. Specificity profiling of human trypsin-isoenzymes. Schilling O, Biniossek ML, Mayer B, Elsässer B, Brandstetter H, Goettig P, Stenman UH, Koistinen H. Biol. Chem. 399 997-1007 (2018)
  14. The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant. Buchholz I, Nagel F, Klein A, Wagh PR, Mahajan UM, Greinacher A, Lerch MM, Mayerle J, Delcea M. Biochim Biophys Acta Proteins Proteom 1868 140281 (2020)


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