NMR Observable-Based Structure Refinement of Membrane Proteins in Explicit Membranes

Abstract

Various NMR observables, such as chemical shift anisotropy (CSA) and dipolar coupling (DC) in solid-state NMR, and NOE-based distance and residual dipolar coupling (RDC) in solution NMR experiments, have been used to characterize membrane protein structures. However, most present membrane protein NMR structure determination approaches refine a protein structure in implicit solvent, and therefore do not provide detailed protein-lipid/detergent interactions, which are essential in determining the relative orientation in the protein's helical domains that is related to its function. In addition, membrane protein NMR observables are often insufficient to clearly define all the side chain-side chain interactions. These undefined interactions could be critical in the protein's structure and function. To overcome these limitations, various NMR observables were utilized as restraints for molecular dynamics (MD) simulations in the explicit (bilayer and micelles) membranes. With various NMR restraint (CSA, DC, NOE and RDC) potentials, we have investigated the orientation of Pf1 coat protein and DAP12-NKG2C complex. The former is a single-pass transmembrane helical protein with a membrane associated periplasmic helix, and the latter is a complex with undefined but functionally required polar side chain interactions. The resulting structures satisfy the NMR observables and compared to the published structures. With respect to the explicit membrane, the disposition of Pf1 TM helix was identified and the dynamic nature of the periplasmic helix orientation was observed. For DAP12-NKG2C complex, several functionally required polar residues adopted different side chain conformations to enhance the complex assembly.