Abstract
We report a high resolution NMR structure and N-15 relaxation studies of the first catalytic cysteine halfdomain (FCCH) of the mouse ubiquitin-activating enzyme El, together with interaction studies of FCCH and the other catalytic El subdomain - SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel 13-barrel structure, a common fold for many proteins with a variety of cellular functions. N-15 relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D H-1/N-15 HSQC spectra of the FCCH. The 2D H-1/N-15 HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain. (C) 2013 Elsevier Inc. All rights reserved.