Faculty Collaboration Database

Identification of a ubiquitin-binding interface using Rosetta and DEER. Proc Natl Acad Sci U S A 2018 Jan 16;115(3):525-530

Date

01/04/2018

Pubmed ID

29295930

Pubmed Central ID

PMC5776994

DOI

10.1073/pnas.1716861115

Scopus ID

2-s2.0-85042128224 (requires institutional sign-in at Scopus site)   23 Citations

Abstract

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.

Author List

Tessmer MH, Anderson DM, Pickrum AM, Riegert MO, Moretti R, Meiler J, Feix JB, Frank DW

Authors

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin
Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Bacterial Proteins
Crystallography, X-Ray
Electron Spin Resonance Spectroscopy
Models, Molecular
Protein Binding
Protein Domains
Pseudomonas aeruginosa
Ubiquitin