The E1/SUMO1-AMSN structure revealed that side chains contacting the Mg ion or ATP β-γ phosphates in E1/SUMO1/ATP·Mg were no longer involved in contacts to the adenylate analog (). Furthermore, the E1~SUMO1-AVSN structure showed that many of these residues were fully displaced from the active site and replaced with residues from the Cys domain during thioester bond formation (). These data suggest that residues required for adenylation should be dispensible for the thioester formation half reaction. The reverse should also hold true. To test this hypothesis, we mutated residues in the SUMO and Ub E1 and assayed these mutant E1s for their ability to form the adenylate, thioester adduct, or tetrahedral intermediate via cross-linking to the Ub/Ubl-AVSN adduct.
Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analog
N-terminal SAE1 helices H1 and H2 are adjacent to the adenylation active site in E1 structures with SUMO1/ATP·Mg and SUMO1-AMSN (). SAE1 Arg21 in helix H2 contacts the ATP γ phosphate and is important for adenylation in other E1s8,10
. A RLW motif in SAE1 composed of Arg 24, Leu 25, and Trp 26 side chains seems to stabilize the positions of the SAE1 H1 and H2 helices through hydrophobic interactions with UBA2 Pro 385, Ile 387, and Tyr 144 side chains. Mutation of Arg21, the RLW motif, or deletion of the N-terminal 27 amino acids of SAE1, which removes helices H1 and H2, abrogated adenylation but had no affect on achieving the closed conformation during thioester bond formation, as evidenced by the mutant E1’s ability to form a cross-link with SUMO1-AVSN (). Thus, side chains within SAE1 amino acids 1–27 are required for adenylation but dispensible for achieving the closed conformation for cross-linking to SUMO1-AVSN, a result consonant with our structure because these elements are fully displaced from the active site in the SUMO1-AVSN structure ().
The UBA2 g1 helix and Lys72 form another surface of the adenylation pocket and residues therein contact the ATP and adenylate ligands in the open conformation (). Individual alanine substitutions for UBA2 Asn56, Leu57, Arg59 and Lys72 resulted in mutants defective for adenylation (). While N56A and L57A mutant E1s readily formed cross-links with SUMO1-AVSN, R59A and K72A mutants lost about half of their cross-linking activity. These results are again consistent with our model because the g1 helix melts and is displaced from the active site in the E1/SUMO1-AVSN structure (). Diminished cross-linking activity for R59A and K72A can be explained by their dual roles in coordinating ATP in the open conformation and in stabilizing interactions in the closed conformation. Specifically, Arg59 contacts the ATP γ phosphate in the open conformation but stabilizes the g1 loop in the closed conformation through hydrogen bond and van der Waals interactions with Asn56 and Leu57 (). Lys72 contacts the ribose 3′-OH, a non-bridging oxygen of β-phosphate and is proximal to the oxygen atoms of both Asn56 and Gln60 side chains in the open conformation but maintains hydrogen bonding interactions with the ribose 3′-OH and Asp50 side carboxylate (3.4 Å) in the closed conformation.
Amino acid residues within the SAE1 N-terminal helix and UBA2 g1 helix are highly conserved across evolution in Ub and Nedd8 E1 enzymes (). To test if mutations of the analogous residues in the Ub E1 would block adenylation while maintaining the ability to achieve the closed conformation to form a cross-link with the Ub-AVSN analog, we deleted the N-terminal 27 amino acids from the Ub E1 and made individual alanine substitutions of Leu472 and Arg474 (correspond to UBA2 Leu57 and Arg59, respectively). As predicted, each mutant isoform was unable to catalyze Ub adenylation as evidenced by the inability to form an E1~Ub thioester (, top). In contrast, each mutant was active in cross-linking assays with Ub-AVSN, suggesting these mutations do not prevent the Ub E1 from achieving the closed conformation during thioester bond formation (, bottom).
We next turned our attention to residues conserved in SUMO, Ub and Nedd8 E1s that appeared important for achieving or stabilizing the closed conformation during thioester formation. The loop that contains the active site cysteine is coordinated by hydrogen bond interactions between the UBA2 Asp50 side chain and backbone amide atoms of Asn177 and Thr178 (). Asp50 is conserved across evolution but is exposed to solvent in E1 structures in the open conformation (). Alanine substitution of Asp50 in SUMO E1 abrogated cross-linking activity with SUMO1-AVSN and thioester bond formation with SUMO1 but had no detectable effect on adenylation. The conservative glutamate substitution (D50E) had no effect on adenylation activity and retained minimal thioester formation and cross-linking activity (). Alanine substitution of the analogous aspartic acid in the Ub E1 also blocked thioester formation and cross-linking to Ub-AVSN (). Thus, Asp50 is essential for maintaining a productive closed conformation during thioester bond formation.
Side chains required for formation of the thioester bond or the tetrahedral intermediate analog are dispensible for adenylation
The UBA2 Arg176 side chain projects into the active site in the closed conformation where it participates in bipartite salt-bridging interactions with Asp117 (2.7 Å and 3.1 Å; ). In the open conformation, Arg176 interacts with the g4 helix in the Cys domain while Asp117 plays an essential role in adenylation by coordinating the magnesium ion in the ATP·Mg complex (). Alanine substitution of UBA2 Arg176 resulted in a slight defect in cross-linking activity and thioester bond formation while maintaining nearly wild-type adenylation activity (), while mutation of Ub E1 Lys596 resulted in no apparent defect in cross-linking or thioester formation (). In contrast, UBA2 D117A and Ub E1 D537A blocked adenylation and abrogated cross-linking activity to low levels (). We hypothesized that the remaining charged side chain in either single mutant might be rescued by eliminating the unpaired charge in the double mutant. As predicted, both UBA2 D117A/R176A or UBA1 D537A/K596A double mutants rescued wild-type activity in the cross-linking assay ().
The cross-over loop and re-entry loops alter conformations in the open and closed forms of the E1 by 90 degrees or more (). To determine if conformational changes in the cross-over loop impacted E1’s ability to catalyze adenylation or thioester formation, we made UBA2 K164A, P165G, T166V, R168P, F170A, P171A, I175A, and N177D substitutions. No single point mutant exhibited significant defects in adenylation, thioester formation, or cross-linking (not shown), consistent with conformational changes occurring over several residues in the cross-over loop. In contrast, conformational changes are localized between two amino acids, Gly381 and Asn382, in the re-entry loop. N382P and G381P/N382P diminished or abrogated thioester formation with SUMO1 or cross-linking to SUMO1-AVSN, respectively, without reduction in adenylation activity (). The single point mutation corresponding to UBA2 N382P in the Ub E1 (K850P) also abrogated cross-linking activity and thioester formation (). These data are consistent with our structure and suggest that conformational changes observed for the closed conformation of the SUMO E1 are important for E1 activity.