Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of...

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.