Structural and biochemical analysis of UBA5 has revealed insight into a structurally minimalistic E1 that does not contain canonical FCCH or SCCH domains. E1 enzymes are thought to perform their multiple activities and interactions through the dynamic action of several modular domains, which are connected by flexible linkers that can undergo extensive structural rearrangements (1
). Similar to other E1 enzymes, the results from the binding assay demonstrate that the CTD of UBA5 also appears to be required for interactions with its E2 enzyme, UFC1. The E1 C-terminal UBL domain is conserved in all E1 enzymes that transfer UBLs to E2-conjugating proteins, and our binding studies of UBA5 also show a dependence on the CTD for thioester transfer of UFM1 to UFC1.
UBL binding specificity and stabilization has been shown to be mediated by a conserved β-sheet in the adenylation domain and also through polar interactions with the FCCH domain (28
). Because native UBA5 does not contain the FCCH domain, we looked for other structural factors that may contribute to UBL substrate specificity or stabilization. In this context, it is interesting to note the presence of the conserved Pro293
residue, which results in the loss of a hydrogen bond between the two final β-strands of the β-sheet that forms part of the UBL-binding surface of the adenylation domain. Further experiments will be required to delineate residues that play important roles in stabilizing interactions with UFM1, while also acting to discriminate against other UBL molecules.
The SCCH domain contains the catalytic cysteine in canonical E1 enzymes and varies in size from ~80 residues in UBA3, ~220 residues in UBA2, and ~265 residues in UBA1. The evolution of the SCCH domain in E1 enzymes has been proposed to be an adaptation to promote reactions with UBLs and E2 enzymes or to allow binding of two UBL molecules simultaneously (A-site and T-site) (14
). For UBA5, the position of the catalytic cysteine in the long α6-helix places it in a position that projects away from the rest of the molecule and would thus likely also allow the simultaneous binding of a UBL to both the A-site and T-site.
Formation of the thioester bond between E1 enzymes and their respective UBL proceeds through the nucleophilic attack of the E1 active site cysteine on the adenylated UBL, with AMP acting as a leaving group. Although it is generally accepted that the E1 catalytic cysteine must be in a deprotonated state to form the thioester bond, there has been little evidence of factors that enhance the cysteine nucleophilicity (30
). A conserved threonine residue that is found in several E1 structures immediately following the catalytic cysteine may contribute to cysteine reactivity and mutation of the residue to an alanine has been shown to greatly reduce thioester bond formation without affecting adenylation of NEDD8 (28
). This threonine is not conserved in UBA5, and the equivalent residue is in fact an alanine for this enzyme. Mutation of residues Arg223
in UBA3 have also been shown to reduce the efficiency of thioester bond formation for this enzyme (14
). However, because these residues are in the SCCH domain of UBA3, a structurally analogous mechanism is not likely for UBA5. Catalytic cysteines are often found at or near the N terminus of α-helices, and the positive charge dipole in this region of various other enzymes has been shown to contribute to a decrease in cysteine pKa
). For example, active site cysteines of the thioredoxin fold family of proteins are commonly found at the N termini of α-helices, and this at least partially accounts for the dramatically lower reported pKa
of these cysteines to as low as 3.0 (35
). Other studies on the influence of helix dipole effects have shown that the position of cysteines at or near the N terminus of α-helices can dramatically affect cysteine pKa
). For example, cysteines introduced into the H helix of myoglobin demonstrated that helix dipole effects alone could account for the pKa
reduction of cysteines at (−ΔpKa
2.1) or near the N-terminal position (−ΔpKa
0.5) in the helix (34
). Another study with helical peptides showed that cysteines two or three residues from the N terminus demonstrated decreases in pKa
of 1.6 and 1.5 pH units, respectively, whereas a cysteine at the N-terminal position demonstrated a pKa
decrease of 1.1 pH units (33
). The unique location of UBA5 Cys250
near the N terminus of the long α6-helix thus also suggests that helix dipole effects at least partially account for the nucleophilic character of this residue. While other E1 enzymes likely employ different mechanisms to enhance cysteine nucleophilicity, the potential need for UBA5 to utilize helix dipole effects may be a consequence of the smaller size of this enzyme when compared with other E1 enzymes.
In all known E1 structures, the catalytic cysteine thiols are >15 Å away from the terminal glycine of their respective A-site bound UBLs (b). This suggests that juxtaposition of the catalytic cysteine and the C terminus of the UBL would require either a major conformational change by the E1 enzyme, displacement of the reaction intermediates, or a combination of both mechanisms. Although it is possible that the adenylated UFM1 C terminus undergoes some displacement between the phosphoester transition state and the thioester transition state, it is also plausible that the Cys250-containing helical segment partially remodels toward this intermediate. Accordingly, the catalytic cysteine of UBA5 is located near a kink in the α6-helix, which is likely to affect the strength of the helix dipole, but could also afford the residue some structural mobility. With this knowledge, structural rearrangements that affect the pKa of the active site cysteine could be used as a mechanism to modulate nucleophilicity to enhance thioester bond formation and transfer reactions.