The free energy landscape of the oncogene protein E7 of human papillomavirus type 16 reveals a complex interplay between ordered and disordered regions

Predrag Kukic,Giuseppe Mattia Lo Piccolo,Roberta Pierattelli,Michele Vendruscolo,Marcela O Nogueira,Dmitri I Svergun,Isabella C Felli

doi:10.1038/s41598-019-41925-4

Abstract

When present, structural disorder makes it very challenging to characterise the conformational properties of proteins. This is particularly the case of proteins, such as the oncogene protein E7 of human papillomavirus type 16, which contain both ordered and disordered domains, and that can populate monomeric and oligomeric states under physiological conditions. Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful method to study these complex systems, most notably in combination with molecular dynamics simulations. Here we use NMR chemical shifts and residual dipolar couplings as structural restraints in replica-averaged molecular dynamics simulations to determine the free energy landscape of E7. This landscape reveals a complex interplay between a folded but highly dynamical C-terminal domain and a disordered N-terminal domain that forms transient secondary and tertiary structures, as well as an equilibrium between a high-populated (98%) dimeric state and a low-populated (2%) monomeric state. These results provide compelling evidence of the complex conformational heterogeneity associated with the behaviour and interactions of this disordered protein associated with disease.

Highlights

Papillomavirus-related infections represent an ubiquitous threat because of their causative link to a range of different types of cancer, including in particular cervical cancer[1,2]
The structural ensemble was determined using all-atom replica averaged metadynamics (RAM) simulations restrained with experimental measurements, a method that combines the sampling efficiency of metadynamics with the on-the-fly modification of the force field with experimental measurements[25]
To extract information about the structure and dynamics of E7 provided by the measured chemical shifts and residual dipolar couplings (RDCs), we incorporated them as replica-averaged structural restraints in molecular dynamics simulations, using the replica-averaged metadynamics (RAM) method[25,26]

Summary

Introduction

Papillomavirus-related infections represent an ubiquitous threat because of their causative link to a range of different types of cancer, including in particular cervical cancer[1,2]. The structural ensemble was determined using all-atom replica averaged metadynamics (RAM) simulations restrained with experimental measurements, a method that combines the sampling efficiency of metadynamics with the on-the-fly modification of the force field with experimental measurements[25] For this purpose, we have measured HN residual dipolar couplings (RDCs) and complemented them with the previously assigned 15N, 1HN, 13Cα, 13C′ and 13Cβ NMR chemical shifts (BMRB entries 19442 and 26069) as experimental restraints. NMR chemical shifts and RDCs are simple population-weighted observables averaged over all conformers that interconvert on timescales faster than the millisecond As such, they can provide information about the dynamics of structural fluctuations of proteins enabling the determination of the structural ensembles that they populate[20]. The combined use of these experimental data enabled us to obtain a converged sampling in the metadynamics simulation and to characterise major structural features of the high-risk HPV-16 E7 in the context of the full-length protein

Methods

Results

Discussion

Conclusion