Wild-type activation complexes containing an E1, Ubl(T), Ubl(A), and an E2 are not stable, because Ubl(T) is readily passed from the E1’s catalytic cysteine to that of E2. Although an E2’s catalytic cysteine is also essential for long-range allosteric changes in E1 structure22
, it was necessary to use a catalytically-inactive E2 harboring a cysteine-to-alanine mutation to trap an activation complex that would provide insights into intermolecular interactions among all the components. Using this approach, we determined the crystal structure of a trapped activation complex containing APPBP1–MBP-UBA3 (NEDD8’s heterodimeric E1, with UBA3 fused to the C-terminus of the MBP crystallization tag), two NEDD8s [NEDD8(T) t
hioester-bound to UBA3’s catalytic Cys216, NEDD8(A) noncovalently-associated at the a
denylation active site], MgATP, and a catalytic cysteine-to-alanine (C111A) mutant of Ubc12 (NEDD8’s E2). All MBP contacts are to regions of APPBP1, UBA3, and NEDD8(A) in conformations identical to previous structures, so MBP is not discussed further in the text. This trapped activation complex is referred to hereafter as APPBP1–UBA3~NEDD8(T)–NEDD8(A)–MgATP–Ubc12(C111A) (, Supplementary Table 1, Supplementary Fig. 1
Previous structural studies revealed that APPBP1–UBA3 and other E1s display common modular architectures, with individual domains specifying each activity: an adenylation domain, a catalytic cysteine-containing domain, and a domain structurally resembling ubiquitin (u
omain, ufd) that binds E213,15,17,23
. Ubls, such as ubiquitin and NEDD8, have two regions: an N-terminal globular domain and a flexible C-terminal tail24,25
. NEDD8’s E2, Ubc12, also has two regions: a unique N-terminal sequence, and a catalytic core domain conserved among all E2s17,21,26
. These features are all visible in APPBP1–UBA3~NEDD8(T)–NEDD8(A)–MgATP–Ubc12(C111A), which adopts a compact overall structure. In this complex, the three APPBP1–UBA3 domains pack to generate a large central groove, which cradles the MgATP, both molecules of NEDD8, and Ubc12 substrates together (). A crossover loop connecting the adenylation and catalytic-cysteine domains divides the groove into two clefts that are continuous both below and above the loop. As in previous structures, when viewed facing the E1 catalytic cysteine located centrally above the adenylation domain, NEDD8(A)’s globular domain binds in the right cleft (Cleft 2) with its C-terminal tail extending under the crossover loop to approach ATP’s α-phosphate in the left cleft (Cleft 1)13,20
. Ubc12’s unique peptide-like extension docks in a groove unique to UBA3’s adenylation domain, and Ubc12’s core domain binds UBA3’s ufd17,21
NEDD8(T) is in the center of the complex, with its C-terminus tethered within a channel focused on the thioester-bond (Supplementary Fig. 2
). A network of charged and polar side-chains contacts UBA3’s catalytic Cys and NEDD8(T)’s C-terminus. Mutational analysis shows these residues contributing to APPBP1–UBA3~NEDD8(T) and Ubc12~NEDD8 complex formation. Conservation of this electrostatic network suggests that common mechanisms underlie E1-catalyzed formation of E1~Ubl and E2~Ubl thioester complexes.
Relative to apo and singly-Ubl(A)-loaded E1 structures13,15,20,21
, the APPBP1–UBA3~NEDD8(T)–NEDD8(A)–MgATP–Ubc12(C111A) structure reveals a striking ~120° rotation of the E2-binding ufd (, Supplementary Fig. 3
). The ufd rotation results in remodeling of the APPBP1–UBA3 central groove to accommodate NEDD8(T)’s globular domain above the crossover loop in the middle of the groove, and Ubc12’s core domain in Cleft 1. In the alternative conformation found in previous apo and singly-Ubl(A)-loaded E1 structures, the central portion of the E1 groove is partially occupied by the E1’s ufd13,15,20,21
(). With APPBP1–UBA3 doubly-loaded with two NEDD8 molecules, the thioester-bound NEDD8(T) would clash with UBA3’s ufd in the apo/singly-Ubl(A)-loaded orientation (), suggesting that the ufd conformational change would accompany doubly-NEDD8-loading of APPBP1-UBA3.
The large-scale ufd rotation reorients Ubc12 as compared to previous models such that (1) Ubc12’s catalytic cysteine faces the direction of UBA3’s catalytic cysteine; (2) Ubc12 is adjacent to the thioester-bound NEDD8(T); and (3) Ubc12 can bind two new E2-binding surfaces not present in apo and singly-Ubl(A)-loaded E1 forms. Interactions between doubly-NEDD8-loaded APPBP1–UBA3 and Ubc12 bury 5600Å2.
In the present structure, which represents a trapped rather than a functioning activation complex, there is a ~20Å gap between Ubc12’s residue 111 (here an alanine, but in wild-type Ubc12 the catalytic cysteine) and UBA3’s catalytic Cys216. Although this gap is still greater than the distance required for transfer of NEDD8(T) to Ubc12, this finding is consistent with previous kinetic studies indicating that long-range conformational changes in E1 structure are induced by an E2’s catalytic cysteine22
, which is absent from our structure. Accordingly, a corresponding ~20Å gap in APPBP1–UBA3’s central groove between NEDD8(T) and NEDD8(A) could accommodate further conformational changes to support the transthiolation reaction.