Role of an N-Terminal Site of Ubc9 in SUMO-1, -2, and -3 Binding and Conjugation

Abstract

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.

E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

“…This result is in part due to E2-mediated effects observed in previous studies (44). The time required for complete TDG modification by SUMO-1 was 60 min at 37°C (Fig.…”

RWD Domain as an E2 (Ubc9)-Interaction Module

“…For NMR studies, uniformly 15 N-labeled RWDD3, RWD, and Ubc9 were expressed in modified M9 medium supplemented with trace minerals and minimal essential medium vitamin solution (Life Technologies Inc.), using 15 NH 4 Cl as the sole nitrogen source as described previously (10,11,14). RWDD3 was expressed in inclusion bodies, denatured with 6 M guanidine hydrochloride, and refolded by dialysis against 50 mM Tris buffer (pH 7.5), 500 mM NaCl, and 1 mM DTT prior to purification.…”

“…15 N-Labeled Ubc9 was titrated with unlabeled RWD domain, or 15 N-labeled RWD domain was titrated with unlabeled Ubc9, and then CSP was monitored using 1 H-15 N HSQC spectra (Fig. 2, B and C).…”

Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

“…A, a strict consensus phylogenetic tree summarizes the sequence relationship within the core domain of Arabidopsis E2s. Subgroups are indicated with Roman numerals, as described (23). The tree is rooted to outgroup AtUFC1, the E2 for UFM conjugation.…”