Crystal Structure of Murine/Human Ubc9 Provides Insight into the Variability of the Ubiquitin-conjugating System

Abstract

Murine/human ubiquitin-conjugating enzyme Ubc9 is a functional homolog of Saccharomyces cerevisiae Ubc9 that is essential for the viability of yeast cells with a specific role in the G 2 -M transition of the cell cycle. The structure of recombinant mammalian Ubc9 has been determined from two crystal forms at 2.0 Å resolution. Like Arabidopsis thaliana Ubc1 and S. cerevisiae Ubc4, murine/human Ubc9 was crystallized as a monomer, suggesting that previously reported hetero-and homointeractions among Ubcs may be relatively weak or indirect. Compared with the known crystal structures of Ubc1 and Ubc4, which regulate different cellular processes, Ubc9 has a 5-residue insertion that forms a very exposed tight ␤-hairpin and a 2-residue insertion that forms a bulge in a loop close to the active site. Mammalian Ubc9 also possesses a distinct electrostatic potential distribution that may provide possible clues to its remarkable ability to interact with other proteins. The 2-residue insertion and other sequence and structural heterogeneity observed at the catalytic site suggest that different Ubcs may utilize catalytic mechanisms of varying efficiency and substrate specificity.Conjugation of ubiquitin to various eukaryotic cellular proteins regulates their activities by controlling protein concentration through ubiquitin-directed degradation (1-4) or by directly modifying protein function through the attached ubiquitin molecules (5). The formation of ubiquitin-protein conjugates proceeds via a cascade of reactions that involve two, or often three, enzymes: the ubiquitin-activating enzyme E1, 1 the ubiquitin-conjugating enzyme Ubc (E2), and the ubiquitin-ligating enzyme E3 (6 -8). The first step is the ATP-assisted formation of a high energy thioester bond between ubiquitin and E1. Ubiquitin is then transferred to a conserved cysteine group of Ubc. In some ubiquitin pathways, Ubc alone, or in cooperation with E3, attaches ubiquitin to the ⑀-amino group of a lysine residue of a substrate via an isopeptide bond. In others, Ubc first passes ubiquitin to a thiol group of E3, and then E3 attaches it to the substrate. Repeated conjugation of ubiquitin to lysine residues of previously bound ubiquitin moieties is required for proteolysis of the substrates by the 26 S proteasome (9). A large body of genetic and biochemical evidence indicates that the Ubcs, together with E3s, are the primary determi-

An NMR-based Model of the Ubiquitin-bound Human Ubiquitin Conjugation Complex Mms2·Ubc13

“…An examination of high resolution E2 structures has revealed that the active site is part of an unstructured loop (41)(42)(43)(44)(45)(46)(47). Our previous and present findings suggest that the interaction of hMms2 with hUbc13 alters the activity of hUbc13 by altering the conformation of the hUbc13 active site.…”

Identification of a Substrate Recognition Site on Ubc9

“…Calculations of Electrostatic Potentials-The surface electrostatic potentials for Ubc9 were calculated using the DelPhi module of IN-SIGHTII (MSI, Inc.) and the crystal structure of Ubc9 (21,24). The solvent dielectric constant was set to 80.…”

lin-35/Rb andubc-18, an E2 ubiquitin-conjugating enzyme, function redundantly to control pharyngeal morphogenesis inC. elegans

“…2 and data not shown). This region of the protein facilitates interactions of E2 enzymes with E1 ubiquitinactivating enzymes (Haas and Siepmann, 1997;Tong et al, 1997) and may play a minor role in interactions with some E3 ubiquitin ligases (Huang et al, 1999;Zheng et al, 2000;Martinez-Noel et al, 2001). A failure to interact with E1 (or with E3) enzymes would be predicted to result in a strong loss of function in this E2, or could possibly lead to an E2 with dominant-negative activity (Townsley et al, 1997;Yamanaka et al, 2000) (however, see previous discussion concerning haploinsufficiency).…”

“…The nature of the molecular lesion identified in ubc-18(ku354) mutants, as well as our RNAi analysis, suggests that ku354 may represent a strong loss-of-function allele. The alteration in chemical charge resulting from the E10K mutation occurs in a region that is required for association with E1 activating enzymes (Haas and Siepmann, 1997;Tong et al, 1997). Several conserved basic residues in this region (equivalent to amino acids R5, R6, and K9 in UBC-18) have been identified as biochemically important for interactions with E1 enzymes (A.…”