title: Expression in Escherichia coli of cysteine protease inhibitors from cowpea (Vigna unguiculata): The crystal structure of a single-domain cystatin gives insights on its thermal and pH stability.
abstract: Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.
authors.affiliation: Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará (UFC), Fortaleza, CE, Brazil.
authors.forename: José Edvar
authors.initials: JE
authors.lastname: Monteiro Júnior
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil.
authors.forename: Napoleão Fonseca
authors.initials: NF
authors.lastname: Valadares
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Brazil.
authors.forename: Humberto D'Muniz
authors.initials: HD
authors.lastname: Pereira
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Brazil.
authors.forename: Fábio Henrique
authors.initials: FH
authors.lastname: Dyszy
authors.affiliation: Departamento de Física, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
authors.forename: Antônio José
authors.initials: AJ
authors.lastname: da Costa Filho
authors.affiliation: Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.
authors.forename: Adriana Ferreira
authors.initials: AF
authors.lastname: Uchôa
authors.affiliation: Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil.
authors.forename: Adeliana Silva
authors.initials: AS
authors.lastname: de Oliveira
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, UFC, Fortaleza, CE, Brazil.
authors.forename: Cristina Paiva
authors.initials: CP
authors.lastname: da Silveira Carvalho
authors.affiliation: Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará (UFC), Fortaleza, CE, Brazil. Electronic address: tbgrangeiro@gmail.com.
authors.forename: Thalles Barbosa
authors.initials: TB
authors.lastname: Grangeiro
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015891:Cystatins / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000072417:Protein Domains; D017421:Sequence Analysis; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins; D014867:Water
keywords: Domain-swapped dimer; Phytocystatin; Recombinant