Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern.

Abstract

The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.