Characterization of the human Retinoblastoma tumor suppressor AB domain in solution

CHEMES, L., GARCÍA ALAI, M.M., ALONSO, L. AND PRAT GAY, G. DE

Trieste, Italia.

Congreso; ICGEB DNA Tumour Virus Meeting,; 2007

ICGEB

The most agreed primary event of HPV E7 proteins required for cellular transformation is the ability to promote the degradation of the retinoblastoma (Rb) tumor suppressor protein in vivo causing 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 proteasomal-mediated degradation of Rb is not clear. Therefore, we are interested in the analysis of 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 with a 9mer peptide containing the LXCXE motif, the E7(1-40) domain and the E7 dimer, yield a clear fluorescence change that allows a titration of the binding interaction and anticipates 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.