Nucleotide-and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

• 成果类型: Article
• 署名作者: Mahajan, Shivansh; Pall, Ashley E.; Li, Yancheng E.; Stemmler, Timothy L.; Rees, Douglas C.; Clemons Jr, William M.
• 署名单位: California Institute of Technology; Wayne State University; Howard Hughes Medical Institute; Chan Zuckerberg Initiative (CZI); Medical University of South Carolina
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9171
• DOI: 10.1073/pnas.2506440122
• 发表日期: 2025-08-29
• 关键词: leptospirillum-ferriphilum; catalytic subunit; binding-site; protein; MODEL; nitrogenase; complex; metalloactivation; hydrolysis; EVOLUTION

摘要

Arsenite (AsIII) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important AsIII resistance mechanism in prokaryotes involves proteins encoded by the ars operon. A central component of the ars operon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of AsIII by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-bound open states, as well as a distinct closed MgATP-bound state liganded to AsIII. X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of AsIII to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by AsIII. This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.