Abstract
E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9–RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.
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Acknowledgements
Use of the APS is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. Use of the SGX Collaborative Access Team beamline facilities at Sector 31 of the APS was provided by Structural GenomiX, who constructed and operates the facility. Use of the NE-CAT beamline at Sector 24 is based upon research conducted at the Northeastern Collaborative Access Team beamlines of the APS, which is supported by award RR-15301 from the National Center for Research Resources at the US National Institutes of Health. Beamline X4A at the National Synchrotron Light Source is supported by the New York Structural Biology Center. We thank M. Gunner for assistance in application of the program MCCE, F. Mohideen for assistance and A. Capili and D. Reverter for critical reading of the manuscript. A.A.Y. and C.D.L. were supported in part by US National Institutes of Health grant GM65872. C.D.L. acknowledges support from the Rita Allen Foundation.
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A.A.Y. generated the data. A.A.Y. and C.D.L interpreted the data and wrote the manuscript.
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Supplementary information
Supplementary Fig. 1
Time course of E2 thioester formation (PDF 1248 kb)
Supplementary Fig. 2
pH titration analysis of wild-type Ubc9 and mutants (PDF 61 kb)
Supplementary Table 1
Catalytic constants and pK values (PDF 26 kb)
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Yunus, A., Lima, C. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat Struct Mol Biol 13, 491–499 (2006). https://doi.org/10.1038/nsmb1104
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DOI: https://doi.org/10.1038/nsmb1104
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