K11-linked Diubiquitin Exhibits Significant Interdomain Dynamics

Abstract

Polyubiquitin chains signal for a wide array of cellular processes, including proteasomal degradation and DNA repair. We hypothesize that the diversity of signaling outcomes stems from the variability in structural and dynamical properties of polyubiquitin chains linked at one of seven different lysine residues (K6, K11, K27, K29, K33, K48 or K63). K11-linked polyubiquitin chains have been found to play an important regulatory role in cell division[1]. While crystal structures of K11-linked diubiquitin have been solved recently[2,3], significant differences in Ub-Ub orientation and arrangement between the two structures suggest interdomain flexibility. Here we characterize the solution properties of K11-linked diubiquitin using NMR spectroscopy. Residual dipolar couplings, standard 15N relaxation measurements, and paramagnetic relaxation enhancements (PRE) all suggest significant interdomain dynamics, even though the individual ubiquitins behave as rigid monomeric ubiquitin entities. PRE data suggest that K11-linked diubiquitin can form a hydrophobic Ub-Ub interface for at least part of the time, similar to the Ub-Ub interface in K48-linked diubiquitin. Titration studies suggest that K11-linked diubiquitin interacts with ubiquitin-binding domains with intermediate affinity between that of K48-linked and K63-linked diubiquitin. Our solution data strongly suggest that K11-linked diubiquitin exhibits structural and dynamical properties different from either K48-linked or K63-linked diubiquitin. 1. Williamson, A. et al. PNAS 106, 18213-18218 (2009). 2. Bremm, A., Freund, S.M.V. & Komander, D. Nat Struct Mol Biol 17, 939-947 (2010). 3.Matsumoto, M.L. et al. Molecular Cell 39, 477-484 (2010).