title: Mapping the substrate binding site of the prostaglandin transporter PGT by cysteine scanning mutagenesis.
abstract: We have identified a cDNA, PGT, that encodes a widely expressed transporter for prostaglandin (PG) E(2), PGF(2alpha), PGD(2), 8-iso-PGF(2alpha), and thromboxane B(2). To begin to understand the molecular mechanisms of transporter function, we have initiated a structure-function analysis of PGT to identify its substrate-binding region. We have found that by introducing the small, water-soluble, thiol-reactive anion Na(2-sulfonatoethyl)methanethiosulfonate (MTSES) into the substrate pathway, we were able to cause inhibition of transport that could be reversed with dithiothreitol. Importantly, co-incubation with PGE(2) protected PGT from this inhibition, suggesting that MTSES gains access to the aqueous pore pathway of PGT to form a mixed disulfide near the substrate-binding site. To identify the susceptible cysteine, we mutated, one at a time, all six of the putative transmembrane cysteines to serine. Only the mutation of Cys-530 to serine within putative transmembrane 10 became resistant to inhibition by MTSES. Thus, Cys-530 is the substrate-protectable, MTSES-inhibitable residue. To identify other residues that may be lining the substrate-binding site, we initiated cysteine-scanning mutagenesis of transmembrane 10 using Cys-530 as an entry point. On a C530S, MTSES-resistant background, residues in the N- and C-terminal directions were individually mutated to cysteine (Ala-513 to His-536), one at a time, and then analyzed for MTSES inhibition. Of the 24 cysteine-substituted mutants generated, 6 were MTSES-sensitive and, among these, 4 were substrate-protectable. The pattern of sensitivity to MTSES places these residues on the same face of an alpha-helix. The results of cysteine-scanning mutagenesis and molecular modeling of putative transmembrane 10 indicate that the substrate binding of PGT is formed among its membrane-spanning segments, with 4 residues along the cytoplasmic end of helix 10 contributing to one surface of the binding site.
authors.affiliation: Renal Division, Department of Medicine, Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. bchan@aecom.yu.edu
authors.forename: B S
authors.initials: BS
authors.lastname: Chan
authors.forename: J A
authors.initials: JA
authors.lastname: Satriano
authors.forename: V L
authors.initials: VL
authors.lastname: Schuster
mesh_terms: D017920:Antiporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008698:Mesylates; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D004268:DNA-Binding Proteins; D008698:Mesylates; D027361:Organic Anion Transporters; C503526:SLCO2A1 protein, human; C109153:(2-sulfonatoethyl)methanethiosulfonate; D015232:Dinoprostone; D003545:Cysteine