title: Effects of cyclization on conformational dynamics and binding properties of Lys48-linked di-ubiquitin.
abstract: In solution, Lys48-linked di-ubiquitin exists in dynamic equilibrium between closed and open conformations. To understand the effect of interdomain motion in polyubiquitin chains on their ability to bind ligands, we cyclized di-ubiquitin by cross-linking the free C terminus of the proximal ubiquitin with the side chain of residue 48 in the distal ubiquitin, using a chemical cross-linker, 1,6-Hexane-bis-vinylsulfone. Our NMR studies confirm that the cyclization affects conformational dynamics in di-ubiquitin by restricting opening of the interface and shifting the conformational equilibrium toward closed conformations. The cyclization, however, did not rigidly lock di-ubiquitin in a single closed conformation: The chain undergoes slow exchange between at least two closed conformations, characterized by interdomain contacts involving the same hydrophobic patch residues (Leu8-Ile44-Val70) as in the uncyclized di-ubiquitin. Lowering the pH changes the relative populations of these conformations, but in contrast with the uncyclized di-ubiquitin, does not lead to opening of the interface. This restriction of domain motions inhibits direct access of protein molecules to the hydrophobic patch residues located at the very center of the interdomain interface in di-ubiquitin, although the residual motions are sufficient to allow access of small molecules to the interface. This renders di-ubiquitin unable to bind protein molecules (e.g., UBA2 domain) in the normal manner, and thus could interfere with Ub(2) recognition by various downstream effectors. These results emphasize the importance of the opening/closing domain motions for the recognition and function of di-ubiquitin and possibly longer polyubiquitin chains.
authors.affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
authors.forename: Ranjani
authors.initials: R
authors.lastname: Varadan
authors.forename: David
authors.initials: D
authors.lastname: Fushman
mesh_terms: D019759:Carbon-Nitrogen Lyases / Q000737:chemistry; D003500:Cyclization; D024202:Electrophoretic Mobility Shift Assay; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry; D014452:Ubiquitins / Q000737:chemistry*
chemical_list: D012997:Solvents; C470301:UBA2 protein, human; D014452:Ubiquitins; C075986:diubiquitin conjugate; D025821:Polyubiquitin; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D044764:Ubiquitin-Activating Enzymes; D008239:Lysine