Identification of a ubiquitin-binding interface using Rosetta and DEER
| Autor: | Jimmy B. Feix, Jens Meiler, Rocco Moretti, David M. Anderson, Dara W. Frank, Adam M. Pickrum, Maxx H. Tessmer, Molly O. Riegert |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 0301 basic medicine Ubiquitin binding Phospholipase Crystallography X-Ray medicine.disease_cause Cofactor 03 medical and health sciences Bacterial Proteins Protein Domains Ubiquitin medicine chemistry.chemical_classification Multidisciplinary biology Pseudomonas aeruginosa Chemistry Electron Spin Resonance Spectroscopy Biological Sciences Protein structure prediction In vitro 030104 developmental biology Enzyme Biochemistry biology.protein Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 115:525-530 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1716861115 |
| Popis: | ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa. The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein–protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron–electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins. |
| Databáze: | OpenAIRE |
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