Structural dissection of a gating mechanism preventing misactivation of ubiquitin by NEDD8's E1 - PubMed (original) (raw)

. 2008 Aug 26;47(34):8961-9.

doi: 10.1021/bi800604c. Epub 2008 Jul 25.

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Structural dissection of a gating mechanism preventing misactivation of ubiquitin by NEDD8's E1

Judith Souphron et al. Biochemistry. 2008.

Abstract

Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL's C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin's Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8's heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin's E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8's residue 72 and UBA3's residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3's preference for NEDD8's Ala72 appears to be indirect, due to proper positioning of UBA3's Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin's Arg72 and an E1's Gln. However, APPBP1-UBA3's failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3's Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.

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Figures

Figure 1

Figure 1

Sequence conservation at a UBL’s residue 72 and E1 residues corresponding to UBA3’s 190. (A) Sequence alignment of the C-terminal tail region of NEDD8 and ubiquitin (UBIQ) from the following organisms: Hs, human; Mm, M. musculus; AT, A. thaliana; Ce, C. elegans; Dm, D. melanogaster. NEDD8 and ubiquitin residue 72 are highlighted. (B) Sequence alignment of the E1 region containing Arg190 (highlighted) from the UBA3 subunit of NEDD8’s E1 (E1 NEDD8) and the corresponding Gln from the UBA1 ubiquitin E1 (E1 UBIQ). (C) Nomenclature and sequences for NEDD8, ubiquitin, and E1 NEDD8 (APPBP1-UBA3) mutants.

Figure 2

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, and (C) GST-APPBP1-UBA3Arg190Ala with NEDD8Ala72 (wt), NEDD8Ala72Arg, NEDD8Ala72Gln, and NEDD8Ala72Lys, as indicated. (D) Representative sensorgrams (left) and binding curves (right) for ubiquitin binding to GST-APPBP1-UBA3Arg190 (wt), GST-APPBP1-UBA3Arg190Gln, and GST-APPBP1-UBA3Arg190Ala, as indicated.

Figure 3

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 indicated mutants of NEDD8, and 3 μM Ubc12. Reactions were stopped at the indicated times, products were separated by SDS−PAGE and detected by Western blotting with anti-NEDD8 antibodies. (B) Western blots of reactions performed as in panel A, except using the indicated His-NEDD8 variants and His-ubiquitin, and probed with anti-His-tag antibodies.

Figure 4

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 residue 190 is shown in various shades of red; NEDD8’s residue 72 in yellow; nitrogen, blue; and oxygen, light red. (A) Overall superimposition of APPBP1-UBA3Arg190Gln (rose)-NEDD8Ala72Arg (melon, “ubiquitinized”) and APPBP1-UBA3Arg190Ala (maroon)-NEDD8Ala72Arg (chartreuse, “wild-type-opposite”) complexes, with close-up view around the NEDD8 mutant’s Arg72 and UBA3’s residue 190. (B) Close-up view showing NEDD8 mutant Arg72 from APPBP1-UBA3Arg190Gln-NEDD8Ala72Arg (melon) and from APPBP1-UBA3Arg190Ala-NEDD8Ala72Arg (chartreuse), and Arg190 (red) from wild-type APPBP1-UBA3-NEDD8. (C) Close-up view showing Arg190 (violet) and NEDD8 mutant Gln72 from APPBP1-UBA3Arg190 (wt)-NEDD8Ala72Gln, and Arg190 (red) from wild-type APPBP1-UBA3-NEDD8.

Figure 5

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 lines for (A) APPBP1-UBA3-NEDD8 (wild-type), (B) APPBP1-UBA3Arg190Ala-NEDD8Ala72Arg (“wild-type-opposite”), (C) APPBP1-UBA3Arg190Gln-NEDD8Ala72Arg (“ubiquitinized”), and (D) APPBP1-UBA3Arg190 (wt)-NEDD8Ala72Gln (“ubiquitinized-opposite”) complexes. Simulated annealing omit Fo−Fc electron density maps are shown in green mesh, contoured at 3σ over UBA3’s residue 190 and NEDD8’s residue 72 in panels B−D. The maps were calculated using the program CNS (32), after simulated annealing at 2000 K omitting both UBA3’s residue 190 and NEDD8’s residue 72.

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