title: Studies on the DIDS-binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle.
abstract: Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys(38), Lys(45), Lys(282), and Lys(413). K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO(3)(-) groups interact with positively charged Lys(282) together with Lys(45) and/or Lys(413). This positions one DIDS isothiocyanate group close to Lys(38), leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys(38) or Lys(290) of MCT1 and Lys(160) or Lys(164) of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys(38) in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys(38) accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp(302-) and Asp(306+), an ion pair already identified as important for transport and located adjacent to Phe(360), which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.
authors.affiliation: Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 1TD, United Kingdom.
authors.forename: Marieangela C
authors.initials: MC
authors.lastname: Wilson
authors.forename: David
authors.initials: D
authors.lastname: Meredith
authors.forename: Chotirote
authors.initials: C
authors.lastname: Bunnun
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Halestrap
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D004910:Erythrocyte Membrane / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D008958:Models, Molecular; D027501:Monocarboxylic Acid Transporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D027981:Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism; D000099233:Monocarboxylate Transport Protein 1
chemical_list: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D006023:Glycoproteins; D008239:Lysine; D008565:Membrane Proteins; D027501:Monocarboxylic Acid Transporters; D027981:Symporters; D000099233:Monocarboxylate Transport Protein 1; C504567:Emb protein, rat