Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution (original) (raw)

2011, Journal of Biological Chemistry

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins. Ubiquitin (Ub) 3 is a small protein composed of 76 amino acid residues and plays regulatory roles in various cellular events, including cell cycle progression, DNA repair, transcriptional regulation, and apoptosis (1, 2). These Ub regulatory functions are expressed by its modification of target proteins through the formation of isopeptide linkages at the C terminus. Ubiquitination is catalyzed by the sequential action of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub-protein ligase E3. The C-terminal group of Ub can also be linked to another Ub (termed the distal and proximal moieties, respectively) through all seven lysine residues at positions 6, 11, 27, 29, 33, 48, and 63 as well as the N terminus, giving rise to various types of polyUb chains (3-7), which mediate diverse signals determining the fate of ubiquitinated proteins. The best characterized case is the Lys-48-linked polyUb chain that serves as a tag for protein degradation by 26 S proteasomes (1). In the Ub/proteasome-mediated proteolytic pathway, Lys-48-linked polyUb is recognized by several proteins possessing Ub-binding motifs (8, 9). These include the proteasomal subunits S5a/Rpn10 and Rpn13 as well as the Ub receptors hHR23/ Rad23, Dsk2/Dph1, and Ddi1/Mud1. NMR studies reported the interactions with the ubiquitin-associated domain of hHR23A and the ubiquitin-interacting motifs of S5a using Lys-48-linked diUb as a minimal model (10, 11). These studies indicated that hydrophobic surfaces, including Val-70 in both Ub units, are involved in the interactions with these Ub-binding motifs. Conformation of Lys-48-linked diUb has also been characterized in the absence of its binding partners. The crystal structures of Lys-48-linked diUb have been solved for two different crystal forms grown under different crystallization conditions (12, 13). Crystallographic studies showed that Lys-48-linked diUb exhibits a "closed" conformation in which hydrophobic surfaces in both Ub units are packed against each other and shielded from the solvent. Identical Ub-Ub interaction modes have been observed in the crystal structures of an engineered tetraUb (14, 15) in which Lys-48 of the second and fourth Ub units are substituted by thialysine and arginine, respectively, and Gly-76 is deleted in the first Ub, and a cyclic tetraUb (16) in which the C terminus of the first Ub is conjugated to Lys-48 of the fourth Ub. Conformational characterization of Lys-48-linked diUb in solution has also been performed using NMR spectroscopy (17, 18). Fushman and co-workers reported a series of NMR studies