Book of Abstracts: Albany 2007
June 19-23 2007
Solution Structure of the Human Pirh2 RING-H2 Domain and its Role in Self-ubiquitylation and Ubiquitylation of P53
The tumor suppressor protein p53 is important in maintaining genome stability and preventing cancer development. In response to cellular stress, the levels and activity of p53 are regulated by ubiquitylation. Knowledge of the mechanism and regulation of the p53 ubiquitylation pathway may reveal novel routes to potential therapeutics to activate p53 and maximize its growth inhibitory and apoptotic effect in cancers. Like Mdm2, pirh2 is an E3 ligase involved in a negative feedback loop with p53. We have determined the solution structure of the Pirh2 RING domain, which displays a compact ββα fold, a cross-braced zinc-binding motif and a hydrophobic depression on the molecular surface. The overall fold of the Pirh2 RING domain is similar to other RING proteins, including Mdm2, but differs from Mdm2 in its oligomeric state, surface charge distribution, and zinc coordination scheme. Pirh2 was shown to be a less efficient E3 ligase in both autoubiquitylation and ubiquitylation of p53 in comparison to Mdm2 in in vitro ubiquitylation assays. However, Pirh2 and Mdm2 both utilized a similar subset of ubiquitin conjugating enzyme E2s including UBE2D and UBE2E subfamilies to facilitate ubiquitylation. Mapping the interactions of Pirh2 and the bound E2s using NMR revealed conserved E2-E3 contacts and a conformational adjustment in the Pirh2 RING domain. This study provides structural and mechanistic insight into molecular mechanisms of p53 regulation by its E3 ligases.
1Ontario Cancer Institute and Department of Medical Biophysics