| Title: | Crystal structure and physicochemical characterization of a phytocystatin from Humulus lupulus: Insights into its domain-swapped dimer |
| Author(s): | Moura GT; Souza AA; Garay AV; Freitas SM; Valadares NF; |
| Address: | "Laboratorio de Biofisica Molecular, Departamento de Biologia Celular, Universidade de Brasilia, Brasilia, 70910-900, Brazil. Laboratorio de Biofisica Molecular, Departamento de Biologia Celular, Universidade de Brasilia, Brasilia, 70910-900, Brazil. Electronic address: napo@unb.br" |
| Journal Title: | Biochim Biophys Acta Proteins Proteom |
| DOI: | 10.1016/j.bbapap.2020.140541 |
| ISSN/ISBN: | 1878-1454 (Electronic) 1570-9639 (Linking) |
| Abstract: | "Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 degrees C to 85 degrees C and high thermal stability at a wide pH range, with DeltaG(25)'s higher than 12 kcal/mol. At 20 degrees C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 2(1) 2(1) and C 2 2 2(1) at resolutions of 1.80 A and 1.68 A, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity" |
| Keywords: | "Cloning, Molecular Crystallography, X-Ray Cystatins/*chemistry/genetics/metabolism Cysteine Proteinase Inhibitors/*chemistry/metabolism Escherichia coli/genetics/metabolism Gene Expression Genetic Vectors/chemistry/metabolism Hot Temperature Humulus/*chem;" |
| Notes: | "MedlineMoura, Gustavo Trajano de Souza, Amanda Araujo Garay, Aisel Valle Freitas, Sonia Maria de Valadares, Napoleao Fonseca eng Research Support, Non-U.S. Gov't Netherlands 2020/09/19 Biochim Biophys Acta Proteins Proteom. 2021 Jan; 1869(1):140541. doi: 10.1016/j.bbapap.2020.140541. Epub 2020 Sep 15" |
