Domain alternation and active site remodeling are conserved structural features of ubiquitin E1

Abstract

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl mallbiquitin-like difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics.

Crystal structure of a human ubiquitin E1–ubiquitin complex reveals conserved functional elements essential for activity

“…Interestingly, HS4 was partially occupied by the Uba1 inhibitor NSC624206 in a recently determined crystal structure of S. pombe Uba1 (PDB code 5UM6) ( Fig. 7B) (22), and although this interaction was not involved in the inhibitory mechanism of the molecule, it further validates the results of the FTMap analysis. Targeting HS1 and HS2 could be a way to perturb the adenylation and E2-binding activity of hUba1, and with the rapid development of protein-targeting chimeric molecule approaches (33) even HS3 and HS4, which would not appear to have an obvious functional consequence of small molecule binding, could potentially be co-opted for proteolytic targeting of hUba1.…”

“…Studies of the E1 for the Ub-like modifier SUMO have shown that the thioester bond formation involves disassembly of the adenylation active site and an ϳ130°rotation of the SCCH domain that transits the catalytic cysteine and other structural elements required for catalysis of thioester bond formation into the active site (24,25). A similar SCCH domain rotation of ϳ106°w as observed for the S. pombe ortholog of Uba1 (22). Finally, the ubiquitin fold domain, UFD, recruits E2 enzymes in a "distal" conformation and subsequently rotates to a more "proximal" conformation that places the E1 and E2 catalytic cysteines into proximity to facilitate E1-E2 thioester transfer (19).…”

“…Analysis of our hUba1-Ub structure and a comparison with other structures of Uba1, SUMO E1, and Nedd8 E1 provide a clue to the structural Human Ub E1-Ub complex structure basis for this observation. As mentioned above, the SCCH domain of Uba1 likely exists in an equilibrium of open (adenylation active) and closed (thioester bond formation active) conformations (22,25). In the open conformation, the adenylation active site is "assembled" with residues positioned optimally for contacts with ATP⅐Mg, and in the closed conformation the adenylation active site is "disassembled" with the structural elements harboring these ATP-binding residues either being disordered or suboptimally positioned (25).…”

“…Ordering of H1 and H2 is also important because it likely shifts the conformational equilibrium of the SCCH domain toward the open state, since ordering of H1/H2 sterically blocks the SCCH domain from adopting the closed conformation. A recently determined structure of S. pombe Uba1 in which the adenylation active site is disassembled and the SCCH domain has undergone a 106°domain alternation relative to the open conformation (Uba1 SCCH_ALT ) suggests that there is an ensemble of SCCH domain conformations between the open and closed states (22).…”

Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme

“…Disassembly of the adenylation active site and concomitant assembly of the thioester bond formation active site following SUMO adenylation and pyrophosphate release serves as a mechanism to drive the SUMO activation process forward 27 . It is likely that other Ubl E1s undergo similar conformational changes during the course of their catalytic cycles 13 , 27 , 40 .…”