title: Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking.
abstract: RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) alpha helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311-314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1-6 (N6) and TMD7-12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6-C6 and N6-N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.
authors.affiliation: Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
authors.forename: Zhanjun
authors.initials: Z
authors.lastname: Hou
authors.forename: Jianmei
authors.initials: J
authors.lastname: Wu
authors.forename: Jun
authors.initials: J
authors.lastname: Ye
authors.forename: Christina
authors.initials: C
authors.lastname: Cherian
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D058978:Reduced Folate Carrier Protein; D013379:Substrate Specificity
chemical_list: D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human