RESUMEN
Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.
Asunto(s)
Estructura Terciaria de Proteína , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Secuencia de Aminoácidos , Western Blotting , Dominio Catalítico , Evolución Molecular , Humanos , Espectrometría de Masas , Modelos Moleculares , Filogenia , Unión Proteica , Homología de Secuencia de Aminoácido , Electricidad Estática , Propiedades de Superficie , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/clasificación , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The tumor suppressor p53 maintains genome stability and prevents malignant transformation by promoting cell cycle arrest and apoptosis. Both Mdm2 and Pirh2 have been shown to ubiquitylate p53 through their RING domains, thereby targeting p53 for proteasomal degradation. Using structural and functional analyses, here we show that the Pirh2 RING domain differs from the Mdm2 RING domain in its oligomeric state, surface charge distribution, and zinc coordination scheme. Pirh2 also possesses weaker E3 ligase activity toward p53 and directs ubiquitin to different residues on p53. NMR and mutagenesis studies suggest that whereas Pirh2 and Mdm2 share a conserved E2 binding site, the seven C-terminal residues of the Mdm2 RING directly contribute to Mdm2 E3 ligase activity, a feature unique to Mdm2 and absent in the Pirh2 RING domain. This comprehensive analysis of the Pirh2 and Mdm2 RING domains provides structural and mechanistic insight into p53 regulation by its E3 ligases.