Folding of a cyclin box: Link between marginal stability, oligomerization and target binding of the retinoblastoma tumor suppressor AB pocket domain. Implications for misfolding in cancer

CHEMES L.B.; NOVAL, M.G.; SANCHEZ, IE; DE PRAT-GAY, G.

Boston

Conferencia; 27th Annual Symposium of the Protein Society; 2013

Protein Society USA

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in eukaryotes. Despite the fact that Rb inactivation is a hallmark of human cancer, the conformational stability and folding of its domains are unknown to date. Rb harbors the highly conserved cyclin fold, which is also present in TFIIB and Cyclin A. The AB pocket domain presents two conserved protein interaction surfaces targeted by cellular and viral proteins and is the site for most tumorigenic missense mutations, which prompted us to investigate the link between conformational stability and ligand binding in this Rb folding unit. The RbAB domain consists of two sub-domains each containing a cyclin fold core, which associate through the A/B interface. We found that RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure loss and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B sub-domain containing the binding site for the LxCxE motif, and the second to unfolding of the stable A sub-domain. The low thermodynamic stability of RbAB translates into a propensity to rapidly aggregate at 37 °C (T50 = 28 min). Denaturation and thermal aggregation are suppressed by HPV and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. Marginal stability and oligomerization may be conserved for function as a ?hub? protein allowing the formation of multi-protein complexes, which could constitute a robust mechanism for Rb to retain its cell cycle regulatory role throughout evolution. The low intrinsic stability of major structural domains and marginal thermal stability at physiological temperature are features shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multi-target signaling pathways.