Structural modularity 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

Congreso; 3rd Latin American Protein Society Meeting-LAPSM; 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 binds in an extended conformation to the RbAB domain, an interaction which we have shown accounts for 90% of the total binding energy between the high-risk HPV16 E7 protein and RbAB (∆G= -11.2 ± 0.2 kcal/mol)[2]. Kinetic studies show that LxCxE motif binds to RbAB following a two-state route 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 E7N domain shows decreased binding affinity due to a faster dissociation rate (koff= 3.3 ± 0.1 s-1) leading to a shorter-lived complex, while high-risk HPV18 E7N behaves similar to HPV16 E7N. The full-length E7 protein 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]. These studies provide a mechanistic basis for understanding the competition established between HPVE7 and cellular proteins containing the LxCxE motif, which may disrupt the Rb protein 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. Acknowledgments: L.B.C was supported by a Fundación YPF Fellowship and I.E.S and G.d.P.G. are Career Investigators from CONICET, Argentina.