Q2N and S65D Substitutions of Ubiquitin Unravel Functional Significance of the Invariant Residues Gln2 and Ser65
- 143 Downloads
Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications.
KeywordsUbiquitin Ubiquitin structure Ubiquitin function G1 β bulge of ubiquitin Mutations of ubiquitin Structure–function relations in ubiquitin
C.R.P. thanks the University Grants Commission, India, for the research grant. The said author is grateful to Prof. Mark Searle and Prof. Daniel Finley for providing plasmids and strains necessary for the study. The author acknowledges the help received from her students Brinda Panchamia and Mrinal Sharma in the preparation of the manuscript.
- 1.Pagano, M. (1997). Cell cycle regulation by ubiquitin pathway. FASEB Journal, 11, 1066–1075.Google Scholar
- 5.Varshavsky, A. (1997). The ubiquitin system. Trends in Biochemical Sciences, 22, 383–387.Google Scholar
- 19.Vijay-kumar, S., Bugg, C. E., & Cook, W. J. (1987). Structure of ubiquitin refined at 1.8 Å resolutions. Journal of Molecular Biology, 194, 513–544.Google Scholar
- 26.Cox, J. P. L., Evans, P. A., Packman, L. C., Williams, D. H., & Wolfson, D. N. (1993). Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin. Journal of Molecular Biology, 234, 483–492.PubMedCrossRefGoogle Scholar
- 47.Ratnaprabha, C., & Sasidhar, Y. U. (1998). Conformational features of disulfide intact and reduced forms of hen egg white lysozyme in aqueous solution in the presence of trifluoroethanol (TFE): implications for protein folding intermediates. Journal of the Chemical Society Faraday Transactions, 94, 3631–3637.CrossRefGoogle Scholar
- 58.Aviel, S., Winberg, G., Massucci, M., & Ciechanover, A. (2000). Degradation of the Epstein-Barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue. Journal of Biological Chemistry, 275, 23491–23499.PubMedCrossRefGoogle Scholar