Structural Refinement of Membrane Proteins by Restrained Molecular Dynamics and Solvent Accessibility Data

Abstract

We present an approach for incorporating solvent accessibility data from electron paramagnetic resonance experiments in the structural refinement of membrane proteins through restrained molecular dynamics simulations. The restraints have been parameterized from oxygen (ΠO 2) and nickel-ethylenediaminediacetic acid (ΠNiEdda) collision frequencies, as indicators of lipid or aqueous exposed spin-label sites. These are enforced through interactions between a pseudoatom representation of the covalently attached Nitroxide spin-label and virtual “solvent” particles corresponding to O 2 and NiEdda in the surrounding environment. Interactions were computed using an empirical potential function, where the parameters have been optimized to account for the different accessibilities of the spin-label pseudoatoms to the surrounding environment. This approach, “pseudoatom-driven solvent accessibility refinement”, was validated by refolding distorted conformations of the Streptomyces lividans potassium channel (KcsA), corresponding to a range of 2–30 Å root mean-square deviations away from the native structure. Molecular dynamics simulations based on up to 58 electron paramagnetic resonance restraints derived from spin-label mutants were able to converge toward the native structure within 1–3 Å root mean-square deviations with minimal computational cost. The use of energy-based ranking and structure similarity clustering as selection criteria helped in the convergence and identification of correctly folded structures from a large number of simulations. This approach can be applied to a variety of integral membrane protein systems, regardless of oligomeric state, and should be particularly useful in calculating conformational changes from a known reference crystal structure.