When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.
Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10. Group I inhibitors exhibit intriguing taxon specificity: potent arthropod-trypsin inhibitors from this group are almost inactive against vertebrate enzymes. The group I member SGPI-1 and the group II member SGPI-2 are extensively studied inhibitors. SGPI-1 is taxon-selective, while SGPI-2 is not. Individual mutations failed to explain the causes underlying this difference. We deciphered this phenomenon using comprehensive combinatorial mutagenesis and phage display. We produced a complete chimeric SGPI-1 / SGPI-2 inhibitor-phage library, in which the two sequences were shuffled at the highest possible resolution of individual residues. The library was selected for binding to bovine trypsin and crayfish trypsin. Sequence analysis of the selectants revealed that taxon specificity is due to an intra-molecular functional coupling between a surface loop and the Lys10-Trp26 core. Five SGPI-2 surface residues transplanted into SGPI-1 resulted in a variant that retained the "taxon-specific" core, but potently inhibited both vertebrate and arthropod enzymes. An additional rational point mutation resulted in a picomolar inhibitor of both trypsins. Our results challenge the generally accepted view that surface residues are the exclusive source of selectivity for canonical inhibitors. Moreover, we provide important insights into general principles underlying the structure-function properties of small disulfide-rich polypeptides, molecules that exist at the borderline between peptides and proteins.
Szenthe
Borbála
B
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
Patthy
András
A
Gáspári
Zoltán
Z
Kékesi
Adrienna Katalin
AK
Gráf
László
L
Pál
Gábor
G
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea; D002417:Cattle; D020650:Combinatorial Chemistry Techniques; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D019476:Insect Proteins; D019151:Peptide Library; D010455:Peptides; D011480:Protease Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; C113210:LOC126284306 protein, Schistocerca gregaria; C106709:pacifastin