UBA3’s Arg190 Gates against Binding to a UBL with a Basic Side-Chain at Residue 72
In order to quantify effects of mutations on APPBP1-UBA3-NEDD8 interactions, we developed a binding assay using BIACORE (Table , Figure ). In our assay, GST-APPBP1-UBA3 binds NEDD8 with a Kd value of 333 ± 1 nM. Furthermore, a NEDD8 mutant harboring the “ubiquitinizing” Ala72Arg mutant does not display any binding in our assay. Thus, we examined the role of UBA3’s Arg190 in this discrimination. Consistent with previous studies, “ubiquitinizing” GST-APPBP1-UBA3 with an Arg190Gln mutation allows binding to NEDD8Ala72Arg, with a Kd of 1.02 ± 0.1 μM.
SPR-Measured Kd and RU Values for Interactions between APPBP1-UBA3 Position 190 Variants and NEDD8 Position 72 Variants
Figure 2 Surface plasmon resonance analysis of APPBP1-UBA3 binding to UBLs. Representative sensorgrams (left) and binding curves (right) from surface plasmon resonance interaction assays, performed as described in , for (A) GST-APPBP1-UBA3Arg190 (wt), (B) GST-APPBP1-UBA3Arg190Gln, (more ...)
We next asked whether relief from negative repulsion from the E1’s Arg is sufficient to allow binding of NEDD8Ala72Arg, or whether attractive electrostatic interaction with the E1’s Gln is required. A UBA3 Arg190Ala substitution would address this question by removing barriers from the Arg side-chain, without providing opportunities for favorable polar interactions with the UBL Arg. GST-APPBP1-UBA3Arg190Ala binds NEDD8Ala72Arg with a Kd value of 1.66 ± 0.01 μM. The similar Kd values (1.02 ± 0.1 μM v 1.66 ± 0.01 μM) for the Gln and Ala substitutions suggest that UBA3’s Arg190 primarily acts as a gate that negatively selects against a UBL’s Arg72. The UBA3 Arg190Ala substitution also allows binding to a NEDD8 with a Lys substitution at residue 72, although in this case a Gln does appear to impart more positive interactions (3-fold better binding).
UBA3’s Residue 190 and NEDD8’s Residue 72 Do Not Play Equivalent Roles in E1-Ubl Selectivity
The UBA3Arg190Ala-NEDD8Ala72Arg pair essentially swaps Arg and Ala selectivity determinants between E1 and NEDD8. Thus, we asked whether these E1 and UBL positions play equivalent roles in determining binding with a complete set of Ala, Arg, and Gln substitutions at both positions (Table , Figure ). The results reveal consistent, nonequivalent, directional effects. First, the wild-type E1-NEDD8 pair binds best, ~5-fold better than the “opposite” pair in which the identities of UBA3’s residue 190 and NEDD8’s 72 have been swapped [UBA3Arg190Ala and NEDD8Ala72Arg]. Second, the “ubiquitinized” pair (with the UBA3Arg190Gln and NEDD8Ala72Arg substitutions to residues found in ubiquitin’s E1 and ubiquitin) associate with a ~9-fold lower Kd than the “opposite ubiquitinized” pair, wild-type UBA3 (Arg190) and NEDD8Ala72Gln.
Interestingly, both the APPBP1-UBA3 Arg190Gln and Arg190Ala variants preferentially bind to NEDD8Ala72Arg (or NEDD8Ala72Lys), rather than to wild-type NEDD8 (Ala72), or the NEDD8Ala72Gln mutant. This raises the possibility that an Arg serves a function beyond dictating E1-UBL selectivity.
NEDD8’s E1 contains Additional Determinants Selecting against Ubiquitin
Having established UBA3’s residue 190 as gating against a UBL’s Arg72, we asked the extent to which this single interaction establishes APPBP1-UBA3’s selectivity against initial noncovalent binding to ubiquitin. Although binding is too weak for us to measure a Kd within the limits of our assay, some signal is detected for interactions between wild-type GST-APPBP1-UBA3 and ubiquitinArg72Ala, but not for wild-type ubiquitin (Figure D). In a related vein, GST-APPBP1-UBA3 Arg190Gln and Arg190Ala do show evidence of interaction with wild-type ubiquitin. Therefore, although the same trends are observed for mutations allowing APPBP1-UBA3 interaction with “ubiquitinized” NEDD8Ala72Arg, additional residues also contribute to NEDD8 E1’s discrimination against noncovalent binding to ubiquitin.
NEDD8’s E1-E2 Transthiolation with UBLs Harboring Arg at Position 72
Although wild-type ubiquitin is excluded from NEDD8’s E1, a previous study found that NEDD8’s E1 could transfer the “NEDD8ylized” ubiquitinArg72Leu mutant to NEDD8’s E2 (Ubc12), albeit with less efficiency overall than the NEDD8 transthiolation cycle (22
). This result raised the question as to whether specificity was established entirely by noncovalent binding by the E1, or whether the ubiquitin mutation relieves discrimination against an Arg at the UBL’s position 72 by APPBP1-UBA3 and Ubc12 during transthiolation. To address this question, we assayed the different APPBP1-UBA3 mutants for NEDD8Ala72Arg transfer to Ubc12 (Figure ). We observe no detectable activity with wild-type APPBP1-UBA3, consistent with gating against this “ubiquitinizing” mutation. However, both the APPBP1-UBA3 Arg190Gln and Arg190Ala mutants can transfer NEDD8Ala72Arg to Ubc12. Similar results are also observed for ubiquitin, although reactions are less efficient, consistent with the weaker noncovalent binding of ubiquitin to these mutants.
Figure 3 Altered E1 NEDD8 (APPBP1-UBA3)-E2 (Ubc12) transthiolation specificity for UBA3 Arg190 mutants. (A) Time-course of forming the Ubc12−NEDD8 thioester complexes with 100 nM wild-type and indicated mutants of APPBP1-UBA3, 4 μM wild-type and (more ...)
Crystallographic Dissection of Arg, Ala, and Gln at UBA3’s Residue 190 and NEDD8’s Residue 72
Our biochemical data pointed toward a directional gating mechanism by which NEDD8’s E1’s Arg190 influences interaction with a UBL’s residue 72. In order to understand the structural basis underlying this specificity, we determined crystal structures of three mutant APPBP1-UBA3-NEDD8 complexes: “wild-type-opposite” [UBA3Arg190Ala-NEDD8Ala72Arg, 2.85 Å resolution]; “ubiquitinized” [UBA3Arg190Gln-NEDD8Ala72Arg, 3.05 Å resolution]; and “ubiquitinized-opposite” [UBA3Arg190 (wt)-NEDD8Ala72Gln, 2.90 Å resolution]. Details of data and refinement statistics are given in Table . Overall, for all three complexes, the electron density 2Fo-Fc maps were continuous and well-defined over all four copies in the asymmetric units (data not shown). Moreover, the side chains at both positions NEDD8’s E1 residue 190 and NEDD8 residue 72 were clearly present in simulated annealing omit maps (Figure ), generated from a model lacking the residues UBA3’s 190 and NEDD8’s 72. For each mutant complex, the four complexes in the asymmetric unit are similar: “wild-type-opposite”, 0.5Å rmsd, “ubiquitinized”, 0.6 Å rmsd, and “ubiquitinized-opposite”, 0.5 Å rmsd. Therefore, the figures display only one copy for each mutant complex. The overall structures all superimpose well with the prior wild-type APPBP1-UBA3-NEDD8 structure, with rmsds between 0.4 and 0.5 Å.
Data and refinement statistics
Figure 5 Differential APPBP1-UBA3 interactions with NEDD8 for UBA3 residue 190 and NEDD8 residue 72 mutants. Stick-representation close-up views, with UBA3 colored red and NEDD8 colored yellow, nitrogen blue, oxygen light-red, and hydrogen bonds shown as dashed (more ...)
Structures of “Wild-Type-Opposite” and “Ubiquitinized” Complexes: Unlocking UBA3’s Gate against a UBL’s Arg72
In order to understand how UBA3’s Arg190 gates against a UBL’s Arg72, we compared the structures of the previously determined wild-type APPBP1-UBA3-NEDD8 complex, in which UBA3 harbors an Arg, and the “wild-type-opposite” and “ubiquitinized” complexes, in which NEDD8 has an Arg. Strikingly, in all three complexes, the Arg guanidium group is in the same relative location (Figure ). This explains how UBA3’s Arg would gate against a UBL’s Arg72 by clashing and repulsion. The results also show how simply removing UBA3’s Arg190 side-chain with an Ala mutation allows a UBL’s Arg.
Figure 4 Structural basis for UBA3’s Arg190s negative selectivity against a UBL’s Arg72. Superimposition of wild-type (21) and mutant APPBP1-UBA3-NEDD8 structures was performed using least-squares fitting over all atoms in O (33). UBA3’s (more ...)
In all three structures, the Arg makes many favorable contacts (Figure ). In the wild-type complex, Arg190 forms hydrogen bonds with UBA3’s Tyr207 and Thr203, and a salt-bridge with UBA3’s Asp179. These latter two electrostatic interactions are preserved when the Arg comes from the mutant NEDD8. The aliphatic portion of the Arg side-chain also preserves hydrophobic interactions with Tyr207 and Tyr321. In the “ubiquitinized” complex, a hydrogen bond between NEDD8’s mutant Arg72 and UBA3’s mutant Gln190 explains the moderate preference for the “ubiquitinizing” Gln over an Ala at position 190 in UBA3.
Insights into Directionality of the UBA3 190 and UBL 72 gate from the “Wild-Type-Opposite” Structure
We wished to obtain a structural understanding of the directional preference toward residue identity at UBA3’s position 190 and NEDD8’s position 72, revealed from our binding assay. Comparison of the wild-type (UBA3Arg190, NEDD8Ala72) with the “wild-type-opposite” (UBA3 mutant Ala190, NEDD8 mutant Arg72) reveals that swapping the Arg side-chain between UBA3 and NEDD8 removes van der Waals contacts, between UBA3 Arg190s gamma and delta carbons and Asp179’s beta carbon (Figure A-B). Asp179 is responsible for orienting UBA3’s Arg151, which in turn aligns the backbone of UBA3’s Mg2+-binding Asp146 and NEDD8’s C-terminal Gly-Gly motif. Although at the resolution of our structures subtle structural differences are not detectable, we speculate that slight variations in this region influences affinity for NEDD8.
Although Arg-Gln and Gln-Arg electrostatic interactions are observed in both the “ubiquitinized” (UBA3 mutant Gln190, NEDD8 mutant Arg72) and “ubiquitinized-opposite” (UBA3 Arg190, NEDD8 mutant Gln72) complexes, the “ubiquitinized-opposite” (UBA3 Arg190, NEDD8 mutant Gln72) interaction is lower affinity. The Arg190 hydrophobic contact to UBA3’s Asp179 is lost in both of these complexes, as in the “wild-type-opposite” complex (Figure C-D). Moreover, the UBA3 Asp179 - Arg salt-bridge is lost in the “ubiquitinized-opposite”, in which the UBA3 Arg guanidium group has moved away, in order to accommodate and form a hydrogen bond with the NEDD8 mutant Gln side-chain (Figure C).
The structure of the “ubiquitinized-opposite” complex suggests that the relatively higher affinity interaction between wild-type UBA3, with an Arg190, and wild-type NEDD8, with an Ala72 is an indirect effect. Despite the lack of positive electrostatic 190−72 interactions, an Ala at NEDD8’s position 72 allows proper positioning of UBA3’s Arg. By contrast, in order to accommodate a Gln at NEDD8’s position 72, other interactions are disrupted for relocation of UBA3’s Arg190 (Figure C, Figure D).