The Ionization State and the Conformation of Glu-71 in the KcsA K+ Channel

AbstractThe side chain of Glu-71 of the KcsA K+ channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69–77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K+ channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900–2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pKa value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pKa of Glu-71 is shifted by ∼+10 pKa units. These results strongly suggest that Glu-71 is protonated under normal conditions