title: Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?
abstract: Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos CP 62210, Mexico. Electronic address: yaselg@ibt.unam.mx.
authors.forename: Yasel
authors.initials: Y
authors.lastname: Guerra
authors.affiliation: Laboratorio de Biología Computacional y Diseño de Proteínas, Centro de Estudios de Proteínas (CEP), Facultad de Biología, Universidad de La Habana, Cuba.
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Valiente
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, Madrid 28029, Spain.
authors.forename: Tirso
authors.initials: T
authors.lastname: Pons
authors.affiliation: Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AT, Wales, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Berry
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos CP 62210, Mexico. Electronic address: rudino@ibt.unam.mx.
authors.forename: Enrique
authors.initials: E
authors.lastname: Rudiño-Piñera
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D057057:Serine Proteases / Q000737:chemistry* / Q000648:ultrastructure; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D012694:Serine; D014360:Trypsin Inhibitor, Kunitz Soybean; D057055:Aspartic Acid Proteases; D057057:Serine Proteases
keywords: Aspartic protease inhibitors; Bi-functional inhibitors; Kunitz-type STI family inhibitors; Plant protease inhibitors; β-Trefoil fold