Abstact

Bacteria resist toxic arsenite (As(III)) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of arsenite and antimonite reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an 'inward-facing' conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that arsenite resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H(+))-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pK(a) estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H(+)-coupling mechanism. Our findings provide structural insights into metalloid recognition and H(+)/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.