title: Structure of BbKI, a disulfide-free plasma kallikrein inhibitor.
abstract: A serine protease inhibitor from Bauhinia bauhinioides (BbKI) belongs to the Kunitz family of plant inhibitors, which are common in plant seeds. BbKI does not contain any disulfides, unlike most other members of this family. It is a potent inhibitor of plasma kallikrein, in addition to other serine proteases, and thus exhibits antithrombotic activity. A high-resolution crystal structure of recombinantly expressed BbKI was determined (at 1.4 Å resolution) and was compared with the structures of other members of the family. Modeling of a complex of BbKI with plasma kallikrein indicates that changes in the local structure of the reactive loop that includes the specificity-determining Arg64 are necessary in order to explain the tight binding. An R64A mutant of BbKI was found to be a weaker inhibitor of plasma kallikrein, but was much more potent against plasmin, suggesting that this mutant may be useful for preventing the breakup of fibrin and maintaining clot stability, thus preventing excessive bleeding.
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Dongwen
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020 São Paulo-SP, Brazil.
authors.forename: Daiane
authors.initials: D
authors.lastname: Hansen
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Ivan G
authors.initials: IG
authors.lastname: Shabalin
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.affiliation: Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, 13560-970 São Carlos-SP, Brazil.
authors.forename: Debora F
authors.initials: DF
authors.lastname: Vieira
authors.affiliation: Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, 13560-970 São Carlos-SP, Brazil.
authors.forename: Marlon V
authors.initials: MV
authors.lastname: de Brito
authors.affiliation: Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, 13560-970 São Carlos-SP, Brazil.
authors.forename: Ana Paula U
authors.initials: AP
authors.lastname: Araújo
authors.affiliation: Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020 São Paulo-SP, Brazil.
authors.forename: Maria Luiza V
authors.initials: ML
authors.lastname: Oliva
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D036481:Bauhinia / Q000737:chemistry*; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D020842:Plasma Kallikrein / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012639:Seeds / Q000737:chemistry; D040681:Structural Homology, Protein
chemical_list: C434623:BbKI protein, Bauhinia bauhinioides; D005343:Fibrinolytic Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; D020842:Plasma Kallikrein; D005341:Fibrinolysin
keywords: Kunitz inhibitor; crystal structure; kallikrein; β-trefoil