Protonation states of important acidic residues in the central Ca²âº ion binding sites of the Ca²âº-ATPase: a molecular modeling study.

ABSTRACT

The P-type ATPases are responsible for the transport of cations across cell membranes. The sarco(endo)plasmic reticulum Ca²âº-ATPase (SERCA) transports two Ca²âº ions from the cytoplasm to the lumen of the sarco(endo)plasmic reticulum and countertransports two or three protons per catalytic cycle. Two binding sites for Ca²âº ions have been located via protein crystallography, including four acidic amino acid residues that are essential to the ion coordination. In this study, we present molecular dynamics (MD) simulations examining the protonation states of these amino acid residues in a Ca²âº-free conformation of SERCA. Such knowledge will be important for an improved understanding of atomistic details of the transport mechanism of protons and Ca²âº ions. Eight combinations of the protonation of four central acidic residues, Glu309, Glu771, Asp800, and Glu908, are tested from 10 ns MD simulations with respect to protein stability and ability to maintain a structure similar to the crystal structure. The trajectories for the most prospective combinations of protonation states were elongated to 50 ns and subjected to more detailed analysis, including prediction of pK(a) values of the four acidic residues over the trajectories. From the simulations we find that the combination leaving only Asp800 as charged is most likely. The results are compared to available experimental data and explain the observed destabilization upon full deprotonation, resulting in the entry of cytoplasmic K⁺ ions into the Ca²âº binding sites during the simulation in which Ca²âº ions are absent. Furthermore, a hypothesis for the exchange of protons from the central binding cavity is proposed.