Mechanistic Studies of ArsA ATPase and ArsB Transporter of the Bacterial Arsenite Efflux System

Arsenic is a notorious metalloid that contaminates the groundwater in several regions worldwide. The trivalent state of arsenic – arsenite (AsIII) – is the abundant species of arsenic under reducing conditions of subsurface waters, and is readily mobilized in aqueous environments. This exposes organisms to toxic concentrations of the metalloid. AsIII is particularly toxic to living systems due to its ability to form stable polar covalent bonds with exposed thiol groups, thus disrupting protein structure and function. Arsenic detoxification systems such as efflux pumps, exist in most organisms that confer tolerance to toxic concentrations of arsenicals found in their environment. The ars operon in many bacteria and some archaea confers resistance to AsIII via ArsB, an integral membrane transporter and ArsA, a cytoplasmic P-loop ATPase. These proteins, collectively referred to as the 'ArsAB efflux pump', facilitate toxic AsIII export in an ATP-dependent manner. In addition, ArsB can operate by itself as a proton-coupled secondary transport and confer intermediate levels of AsIII resistance. The mechanisms of this dual mode of AsIII efflux are poorly understood, particularly the molecular events associated with the capture of AsIII from the cytoplasm by ArsA, its transfer to ArsB and subsequent vectorial transport across the membrane. Apart from understanding fundamental mechanisms of toxic metalloid detoxification in living systems, molecular-level investigations of AsIII efflux systems are of broad biotechnological interest for their potential to inform robust and sustainable bioremediation strategies. In this thesis, we elucidate the mechanism of ArsA ATPase and ArsB transporter using structural approaches. We characterized the nucleotide hydrolysis mechanism of ArsA by single particle cryogenic electron microscopy (cryo-EM), outlining various conformational states of the ATPase that modulate the nucleotide-dependent capture and delivery of AsIII for efflux. We show that this mechanism is consistent with the general mechanistic framework of the Intradimeric Walker A (IWA) family of ATPases. Furthermore, overexpression and purification of the membrane transporter ArsB enabled characterization of the first structure of ArsB by cryo-EM, in both apo and AsIII-bound states. Lastly, we show that ArsB enhances steady-state ATPase activity of ArsA, indicating a direct interaction between the two components of the efflux pump. Computational modeling gives some insights into a putative ArsAB interaction interface. While several mechanistic questions remain, the findings reported in this thesis together constitute a foundation for future mechanistic elucidation of the ArsAB efflux system.