Lipid dependence of BsYetJ calcium transport activity by fluorescence and ESR techniques.

Abstract

Recent studies based on X-ray crystallography and pulsed electron spin resonance results have revealed substantial structural insights for the mechanistic understanding of the Ca2+ transport mechanism in the membrane protein YetJ from Bacillus subtilis (BsYetJ). One striking finding is that its structure-function relationship is sensitive to the difference between the surrounding environments, detergent versus POPC lipid. Here, we continue on the study to explore the molecular details of BsYetJ in response to changes in lipid composition, at various mixtures of PC, PE, PI, and cholesterol. Using mutagenesis and fluorescence-based functional assays, we investigate several important negatively charged residues in the BsYetJ and reveal how the interplay determines the calcium channel conductance. This study identifies the conformations of BsYetJ in a complex lipid environment using electron spin resonance and molecular dynamics. Together with the functional data, we propose a mechanism, composed of previously unidentified selectivity filters, for the calcium transport activity of BsYetJ. We show that the calcium transport activity of BsYetJ can be modulated by lipid compositions. Our study provides new insights into the structural impact of the mutations and the development of the mechanistic model of BsYetJ.