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 (PO 2) and nickel-ethylenediaminediacetic acid (PNiEdda) 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 spinlabel 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.

Resting state of the human proton channel dimer in a lipid bilayer

“…S5, Bottom), but also in their voltage dependences and apparent slopes of activation (1,35). Our initial CiVSD-WT-based structural model was further refined by EPR constraints using a pseudoatomdriven solvent accessibility refinement method PaDSAR (26,36) that uses experimental solvent accessibilities as soft constraints. The resulting model was equilibrated in explicit lipid POPC for 400 ns and proved to be stable as a representation of the resting state of hHv1-VSD in lipid bilayer.…”

“…The four MD stable models were evaluated with experimental data primarily with ΠNiEdda and NEM/AMS accessibility data. The structural model correlating best with experimental accessibilities was subjected to an additional MD simulation with constraints from present EPR spectroscopic data using an established computational method, PaDSAR (36). The constrained model was equilibrated in explicit lipid POPC for 400 ns, which serves our working model for the resting state of hHv1-VSD.…”

“…A spin label (ONP0) pseudoatom, representing the nitroxide spin label, was covalently attached to the alpha carbon atom of each residue (62,63). Sufficient solvent OXY and NIC pseudoatoms (36), representing O 2 and NiEdda, were introduced into the system and distributed into the membrane and water compartments, respectively. The interactions between pseudoatoms and water; pseudoatoms and ions; and OXY and lipids were turned off.…”

“…The O 2 and NiEdda accessibility data from EPR experiments were used as structural restraints to further refine the hHv1-VSD structural model based on CiVSD-WT with the PaDSAR approach (26,36). Three types of spin-label pseudoatoms (ONP1, ONP2, and ONP3) and three types of environmental pseudoatoms (PROT, OXY, NIC) were introduced into the system.…”

The activated state of a sodium channel voltage sensor in a membrane environment

“…This information was used to evaluate atomic models of NaChBac-VSD through restrained molecular dynamics (MD) simulations incorporating solvent accessibility values as experimental restraints (19). This model is compared with the crystal structures of the VSD of KvAP and Kv1.2-2.1 chimera (6,20) and demonstrates that the general structural principles and basic functional properties of VSD domains are conserved across K þ and Na þ channels.…”