Abstract
Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.
Highlights
S entry into the cell (Kertesz, 2000)
The structure of P. fragi CysZ (PfCysZ) was solved second, to 3.5 Å 102 resolution, with crystals belonging to space group C2
We determined the structure of P. denitrificans CysZ (PdCysZ), which crystallized in multiple forms belonging to space groups P63, P4122 and P212121, revealing the same architecture and oligomeric assembly each time (Fig. 1-S2b)
Summary
S entry into the cell (Kertesz, 2000). In certain fungi, and prokaryotes, once internalized, SO42- is first reduced to sulfite (SO32-), and further to sulfide (S2-), a form that can be used by the cell (Kredich, Hulanicka, & Hallquist, 1979) (Fig. 1-S1). To investigate the role of CysZ in cellular sulfate uptake at a molecular level, we have undertaken an approach that combines structural and functional studies To this end, we determined the crystal structures of CysZ from three species, Idiomarina loihiensis (Il; IlCysZ), Pseudomonas fragi (Pf; PfCysZ), and Pseudomonas denitrificans (Pd; PdCysZ), and characterized CysZ function in purified form, in reconstituted proteoliposomes, in planar lipid bilayers, and in cells. Interpreting the functional data in a structural context has allowed us to formulate a mechanistic model for CysZ-mediated SO42- translocation across the bacterial cytoplasm membrane Both the structures and the functional properties of CysZ proteins are distinct from those of any known membrane transporter or ion channel. These distinctive properties make CysZ appealing as a model system for studies of biophysical principles of membrane protein biogenesis and transmembrane ion passage
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