Selective inhibition of trypsins by insect peptides: role of P6-P10 loop.
PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K(i) around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K(i) <or= 0.02 nM). Therefore, they display a selectivity not existing for the parent chymotrypsin inhibitor PMP-C. By NMR, we demonstrate that HI possesses a highly rigid structure similar to the crystal structure of a variant of PMP-D2 in complex with bovine alpha-chymotrypsin. The main difference with PMP-C is located in the region from residues 20 to 24 (positions P6-P10) that interacts with the loop containing Gly173 in chymotrypsin. The corresponding residue in mammalian trypsins is always a proline that may generate a steric clash with the inhibitor. The residues thought to confer selectivity were mutated with PMP-C as a model. The resulting analogue PMP-D2(K10W,P21A,W25A) loses some activity toward insect and fungal trypsins but is a more potent inhibitor of mammalian trypsins, corresponding to a decrease of selectivity. This work represents a first attempt in tuning the selectivity of natural peptidic serine protease inhibitors by mutating residues out of the reactive loop (P3-P'3).
Kellenberger
C
C
Centre d'Immunologie de Marseille-Luminy, UMR 145, Parc Scientifique et Technologique de Luminy, Case 906, 13009 Marseille, France. kellenberger@ciml.univ-mrs.fr
Ferrat
G
G
Leone
P
P
Darbon
H
H
Roussel
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry; D047169:Cyclotides; D005670:Fusarium / Q000201:enzymology; D006110:Grasshoppers / Q000737:chemistry / Q000201:enzymology; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013552:Swine; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
D047169:Cyclotides; D019476:Insect Proteins; D014361:Trypsin Inhibitors; C072986:PMP-D2 peptide, insect; C072987:PMP-C peptide, insect; D002918:Chymotrypsin