Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding. ACS Omega 2017 Jun 30;2(6):2977-2984
Date
07/12/2017Pubmed ID
28691114Pubmed Central ID
PMC5494639DOI
10.1021/acsomega.7b00349Scopus ID
2-s2.0-85028991891 (requires institutional sign-in at Scopus site) 10 CitationsAbstract
ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, that is, it has multiple thermodynamically stable conformations that interchange depending on its environment. Such flexible proteins pose unique challenges to structural biology: (1) not only is it often difficult to determine structures by X-ray crystallography for all biologically relevant conformations because of the flat energy landscape (2) but also experimental conditions can easily perturb the biologically relevant conformation. The first challenge can be overcome by applying orthogonal structural biology techniques that are capable of observing alternative, biologically relevant conformations. The second challenge can be addressed by determining the structure in the same biological state with two independent techniques under different experimental conditions. If both techniques converge to the same structural model, the confidence that an unperturbed biologically relevant conformation is observed increases. To this end, we determine the structure of the C-terminal domain of the effector protein, ExoU, from data obtained by electron paramagnetic resonance spectroscopy in conjunction with site-directed spin labeling and in silico de novo structure determination. Our protocol encompasses a multimodule approach, consisting of low-resolution topology sampling, clustering, and high-resolution refinement. The resulting model was compared with an ExoU model in complex with its chaperone SpcU obtained previously by X-ray crystallography. The two models converged to a minimal RMSD100 of 3.2 Å, providing evidence that the unbound structure of ExoU matches the fold observed in complex with SpcU.
Author List
Fischer AW, Anderson DM, Tessmer MH, Frank DW, Feix JB, Meiler JAuthors
Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of WisconsinDara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin
