Local and global conformational plasticity of the HPV-16 E2 DNA binding domain,

NADRA, A.D., ELISEO, T., PRAT GAY, G. DE, PACI, M. AND CICERO, D.O.

Alghero, Italy. September 21-24, 2004

Congreso; XXXIV National Congress on Magnetic Resonance; 2004

GIDRM

LOCAL AND GLOBAL CONFORMATIONAL PLASTICITY OF THE HPV-16 E2 DNA BINDING DOMAIN Alejandro D. Nadra‡, Tommaso Eliseo†, Diego U. Ferreiro‡, Maurizio Paci†, Gonzalo de Prat-Gay‡ and Daniel O. Cicero† ‡ Instituto Leloir - Inst. de Investigaciones Bioquímicas, FCEyN-UBA and CONICET, Argentina. †Dipartimento di Chimica, Università di Roma “La Sapienza”, Rome, Italy. E-mail: anadra@leloir.org.ar Gene transcription in papillomavirus is controlled by the E2 protein. We analyzed the structure and dynamics of the high risk strain HPV16 DNA E2 binding domain as a prototypic case. Using NOE constrains and residual dipolar couplings (RDC), we have obtained a high resolution structure and were able to address several issues related to folding and functional particularities of this unique fold. Despite being a highly stable and cooperatively folded dimer (DG ~12.0 kcal/mol), exchange measurements show that over 90% of the protons are exchanged after 12 hrs. Careful backbone dynamics measurements indicated that the flexibility of the DNA recognition helix is particularly high for its N-cap residues. The few residues that remain unexchanged after 24 hrs are I286 and V287 of b-strand 1, I330 and L333 of b-strand 3 and residues N343 and F344 of a-helix 2, and these long range interactions are critical for the overall stability of the fold. Thus, the recognition helix, far from being disordered as found in several transcriptional regulatory domains, acts as a structural hinge, providing an overall plasticity with a stable b-barrel architecture. This plasticity provides potential regulatory conformational changes in regions far from the DNA recognition interface, an essential necessary role in these and other transcriptional regulators. In addition, we have completely assigned the backbone of the HPV16 E2C domain bound to a 18mer DNA duplex containing the specific E2 recognition site. The perturbation of backbone chemical shifts is small, except for the residues in the major DNA binding helix and adjacent residues, and a in the b2-b3 loop. Backbone amide exchange rate shows a larger overall protection but most notably in the DNA binding helix. The C-terminus located oppostite to the DNA binding helix and locking the central b-barrel interface with crytical H-bonds, becomes more protected, supporting a role of these interactions in DNA bending. These results suggest that the overall plasticity of the fold is not restricted to the DNA binding interface.