Dissecting non canonical secondary structural elements within Human Papillomavirus E7 oncoprotein E7N intrinsically disordered domain.

NOVAL, M.G.; CHEMES L.B.; GALLO, M.; SALVAY, A; PERRONE, S.; DE PART-GAY, G.

Boston, MA, USA

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

Protein Society USA

Intrinsically disordered proteins (IDPs) lack a well defined secondary or tertiary structure under physiological conditions. However, they are largely represented in protein-protein interaction networks. These proteins have some local preferences for secondary structure elements in the unbound state favoring their interactions with their protein partners. It has been described that different environmental factors affects the IDP energy landscape, determining their conformational plasticity. We have previously reported that the Human Papilloma Virus Type-16 E7 oncoprotein is an IDP. The E7 protein is an acidic protein with an intrinsically disordered N-terminal domain (E7N) that can bind to at least 40 different target proteins. E7N presents two conserved regions CR1 and CR2 and five short linear motifs (DRYK1A ubiquitination motif, E2F mimic, LxCxE Rb binding motif, CKII and PEST) which are supposed to be responsible for the binding promiscuity of this domain. In this work we aimed to study the conformational plasticity of E7N, using a fragmentation approach in combination with circular dichroism spectroscopy (CD), NMR, analytical ultracentrifugation, and solvent stabilization. By TFE titrations of E7N domain and fragments followed by CD we identified two regions with α-helix propensities, one of them located within the CR1 and the other within the CR2 region. We further confirmed the presence of both α-helixes by NMR. At pH 7.5, we found that the first helix span the segment from L8 to L13 and the second from P17 to N29. We have also identified a region with polyproline type II structure comprising residues from E33 to D39, which is in agreement with CD analysis. At pH 5.0 we found that only the second helix was affected, being propagated from residue 29 to 38 with the concomitant loss of pII structure. Interestingly, the linear motifs identified for this domain are located within the region with fluctuating secondary structure, suggesting that this conformational plasticity may modulate interactions with the different binding partners. In addition, at lower pH and with submiscelar SDS concentration we found that a ß-sheet type structure is stabilized only in E7N, but not in other sub-fragments, indicating that the structure formed is not based on just local information. Altogether these results strongly suggest that in its unbound state, E7N exists as an equilibrium of interconverting structures, exposing interacting linear motifs and thus might determine binding to cellular and viral targets.