Increased stability and DNA binding of monomeric variants of the dimeric DNA binding domain of the human papillomavirus E2C transcriptional regulator

DELLAROLE, M. AND PRAT GAY, G. DE.

Pinamar, Argentina. Diciembre 3-6, 2005

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2005

SAIB

INCREASED STABILITY AND DNA BINDING OF MONOMERIC VARIANTS OF THE DIMERIC DNA BINDING DOMAIN OF THE HUMAN PAPILLOMAVIRUS E2C TRANSCRIPTIONAL REGULATOR Mariano Dellarole & Gonzalo de Prat-Gay Fundación Instituto Leloir E-mail: mdellarole@leloir.org.ar Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The expression of all viral genes is controlled by the E2 transcriptional regulator, which also participates in DNA replication, and the recognition of DNA is carried out by the C-terminal dimerization domain (E2C). In late stages of the infection, E2 represses the expression of the two main oncoproteins E6 and E7, and disruption of the E2 ORF upon insertion in the host genome was proposed as an irreversible step towards cell transformation. In order to test the hypothesis of improving stability and DNA binding affinity of E2C we constructed monomeric variants by inserting a neutral linker between two consecutive E2C genes, generating a monomeric species. The two best expressing variants, with 6 and 12 residue linkers (E2Csc-6 and E2Csc-12), were purified and characterized. Both show overall identical conformation to the natural dimer as judged by CD and fluorescence spectroscopy. Urea denaturation experiments show cooperative transitions with their stabilities largely improved by 4.7 kcal.mol-1 for the E2Csc-12, and 3.0 kcal.mol-1 for the E2Csc-6 residue linker. At 150 mM phosphate, E2Csc-12 displays a KD of 0.5 ± 0.1 nM compared to 2.52 ± 0.3 nM for E2C. Binding of E2Csc-6 shows a deviated salt dependence indicative of differential release of ions upon binding and suggesting a strained conformation compared to the 12 residue linker and E2C. Finally, calorimetric experiments show very similar îH values around 22 kcal.mol-1, further supporting an identical interface to the reference dimer, with similar and low entropic contribution, indicative of a mainly enthalpically driven process. Section: Enzymology and Structural Biology