Molecular basis for phosphorylation dependent PEST mediated protein turnover.

GARCÍA ALAI, M.M., GALLO, M., SALAME, M., WETZLER, D.E., MC BRIDE, A.A., PACI, M., CICERO, D.O. AND PRAT GAY, G. DE.

Pinamar, Argentina. Diciembre 3-6, 2005

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2005

SAIB

Molecular basis for phosphorylation dependent PEST mediated protein turnover. Maria M. García-Alai1, Mariana Gallo2, Marcelo Salame1, Diana E. Wetzler1, Alison A. McBride3, Maurizio Paci2, Daniel O. Cicero2, and Gonzalo de Prat-Gay1, 1 Instituto and UBA, 2 University of Rome ´Tor Vergata, 3Laboratory of Viral Diseases, NIAID, NIH. E-mail: mgarciaa@leloir.org.ar Rapid protein turnover regulates the intracellular levels of proteins involved in key cellular processes. In many cases, proteasomal-mediated degradation of these proteins is modulated by phosphorylation of short PEST sequences. The E2 protein from bovine papillomavirus participates in essential steps in the life cycle of this transforming virus, such as gene transcription, DNA replication and episomal genome maintenance. Phosphorylation of a PEST sequence located in the flexible hinge region of E2 accelerates its degradation, determining the level of viral genome copy number. To investigate the effects of phosphorylation on the conformation and stability of this region, we determined the structure of a p29 amino acid peptide fragment containing the PEST sequence (E2-PEST) by NMR methods. The E2-PEST peptide has pH dependent polyproline II and ±-helix structures, connected by a turn around the actual PEST sequence. The overall structure is disrupted by phosphorylation, in particular that at serine 301, and substitution of different residues within this region can have either stabilizing or destabilizing effects. There is an excellent correlation between the structural stability of different peptides in vitro and the resulting half-life of full-length E2 proteins containing the same mutations in vivo. The structure around PEST region is not unfolded and appears to have evolved to display a marginal stability, finely tunable by phosphorylation or mutations. We propose that conformational stability, rather than recognition of a phosphate modification, modulates the processing of PEST sequences by the proteasome machinery or other proteases. Section: Enzymology and Structural Biology