Structural Basis for Conformatioanl Space of Linear Polyubiquitin by Crystallography and Solution Scattering

Abstract

Conformational flexibility of linkage-specific polyubiquitin chains enables ubiquitylated proteins and their receptors to be involved in a milieu of cellular processes. Linear or M1-linked polyubiquitin chains, associated with non-degradational cellular signalling pathways, have been known to adopt multiple conformations from compact to extended ones. However, the extent of such conformational flexibility remains open. Here we determined the crystal structure of linear Ub2 in a more compact conformation than that of the previously known structure (PDB ID: 3AXC). The two structures differ significantly from each other evidenced by r.m.s.d. among Cα atoms being 3.1 A. The compactness of our linear Ub2 structure in comparison with 3AXC is supported by the smaller values of radius of gyration (Rg, 18 A vs. 18.9 A) and maximum interatomic distance (Dmax, 55.5 A vs. 57.8 A). Extra intramolecular hydrogen bonds formed among polar residues between distal and proximal ubiquitin moieties seem to contribute to the stabilization of the compact conformation of linear Ub2. We also observed an ensemble of three semi-extended and extended conformations of linear Ub2 by small-angle X-ray scattering (SAXS) analysis in solution. In addition, the conformational heterogeneity in linear polyubiquitin chains is clearly manifested by SAXS analyses of linear Ub3 and Ub4: at least three distinct solution conformations are observed in each chain, with linear Ub3 conformations being compact. Our results expand the extent of conformational space of linear polyubiquitin chains and suggest that changes in conformational ensemble may be pivotal in mediating multiple signalling pathways.