Interaction of the HPV E7 protein with the human retinoblastoma AB domain in solution

CHEMES, L.; DE PRAT-GAY, G.

Departamento de Física, FCEN UBA, September 3-7 Buenos Aires Argentina

Workshop; First South American Workshop on Advanced Fluorescence Microscopy Techniques; 2007

The Fluorescence Foundation and Departamento de Física, FCEN UBA

A primary event in HPV-mediated oncogenesis is the interaction of HPV E7 proteins with the retinoblastoma (Rb) tumor suppressor, which leads to destabilization and proteasomalmediated degradation of Rb in vivo. This causes E2F release, with the ensuing consequences on gene transcription of essential S-phase genes. HPV-E7 is known to interact with Rb both in vivo and in vitro, but the exact mechanism by which E7 induces degradation of Rb is not clear. Several domains of both E7 and Rb have been implicated in the high-affinity inter action between these two proteins. Furthermore, E7 proteins of high and low risk strains have been shown to possess differential affinity for Rb. Therefore, we were interested in analyzing the mechanism of interaction between HPV E7 and Rb in solution. Biophysical studies of this interaction have been hindered by the difficulty in obtaining large amounts of Rb protein suitable for characterization. Therefore, we have initially focused on the optimization of Rb AB scaled-up expression and on the study of its properties in solution. We describe the purification to homogeneity of the Rb AB domains from bacteria, which are obtained as a monomeric species of the expected molecular weight (~48 KDa). The protein presents a typical alpha-helical circular dichroism spectrum, as previously described. Rb AB is stable at different pHs with some aggregation taking place in the range between pH 4.0 and 5.0 and surprisingly shows substantial persistent helical structure at pH 2.0. Chemical denaturation by gdm.chloride follows a biphasic tertiary and secondary structure change, suggesting an independent behavior of the domains. The small change in exposition of the trp residue in the first transition is consistent with the pH unfolding the weakest domain, most likely the B domain. However, Rb AB is resistant to urea denaturation, suggesting a role for electrostatic interactions in the stabilisation. We show that interaction of a 9mer peptide containing the LXCXE motif, the E7(1- 40) domain and the E7 dimer with Rb-AB are able to stabilize the protein to thermal denaturation. Preliminary binding studies also anticipate a possible mechanistic, kinetic, and thermodynamic dissection of this key interaction for HPV linked cancers with implications for other DNA tumour viruses with equivalent oncoproteins.