Structural modularity, intrinsic disorder and electrostatic interactions along the kinetic association route of the HPV E7 oncoprotein and the retinoblastoma tumor suppressor

CHEMES, L.B.; SÁNCHEZ, I.E.; DE PRAT-GAY, G.

Salta, Argentina

Simposio; 3rd Latin American Protein Society Meeting-LAPSM-SAB; 2010

The human retinoblastoma tumor suppressor (Rb) is a key regulator of cell cycle progression and chromatin state[1], and is the main target through which HPVE7 controls the host cell proliferation state. The intrinsically disordered N-terminal domain of E7 (E7N) presents an LxCxE motif followed by a highly conserved acidic region containing a CKII phosphorylation site (CKII-PEST). The LxCxE motif of HPV16E7 binds in an extended conformation to the RbAB domain, accounting for 90% of the total binding energy (∆G= -11.2 ± 0.2 kcal/mol)[2]. Kinetic studies show that this motif binds to RbAB following a two-state route and presenting a strong electrostatic component that is present in the transition state ensemble (TSE). Both the CKII-PEST region and its phosphorylation contribute to the interaction and exhibit strong electrostatic dependence, coincident with the presence of complementary charges in both binding surfaces. The CKII-PEST contribution is present in the TSE, presumably due to the establishment of long-range charge-charge interactions. Instead, only part of the contribution due to phosphorylation is present in the TSE. The low-risk HPV11 and the high-risk HPV18 E7N domains show kinetic differences, which are reflected mainly in the dissociation rate. Full-length E7 associates through a multi-state route involving the formation of an encounter complex, followed by conformational rearrangements, in accordance with the previously described presence of more than one binding site within E7[2]. The contribution of the intrinsically disordered nature of E7 to Rb targeting will be discussed, integrating experimental data with an analysis of the variability of motif features in PV E7 proteins. The comparison of the motifs present in viral and host proteins that bind to Rb and of their affinities provides a better understanding of the competition established between viral and cellular proteins interacting with Rb, which in certain cases leads to disruption of the Rb interaction network. 1. Weinberg, R.A. Cell, 1995. 81(3): p. 323-30.; 2. Chemes, L.B., et al. FEBS J, 2010. 277(4): p. 973-88.