Abstract
All extant life forms require trace transition metals (e.g., Fe2/3+, Cu1/2+, and Mn2+) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.
Highlights
Owing to the limited chemical reactivity of amino acid side chains, metals such as Cu1/2+, Zn2+, Mn2+, and Fe2/3+ are indispensable cofactors for the structure and function of many enzymes and structural proteins, and it is estimated that metalloproteins comprise 30 to 40% of most proteomes [1, 2]
ABC transporters that function as importers depend on a high-affinity substrate-binding protein (SBP) that delivers the substrate to the cognate transporter [21,22,23]
Substrate binding by the SBP is essential for transport [22, 23, 29, 30], it is not sufficient: several studies have established that the SBP may bind ligands that are not transported by the transporter [31,32,33]
Summary
Received for publication, February 9, 2021, and in revised form, August 12, 2021 Published, Papers in Press, August 18, 2021, https://doi.org/10.1016/j.jbc.2021.101087 Anastasiya Kuznetsova1,‡, Gal Masrati2,‡ , Elena Vigonsky1, Nurit Livnat-Levanon1, Jessica Rose1, Moti Grupper3, Adan Baloum1, Janet G. Yang4 , Douglas C. Rees5, Nir Ben-Tal2, and Oded Lewinson1,* From the 1Department of Molecular Microbiology and the Rappaport Institute for Medical Sciences, Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel; 2Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel; 3Infectious Disease Unit, Rambam Health Care Campus, Haifa, Israel; 4Department of Chemistry, University of San Francisco, San Francisco, California, USA; 5Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA
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