Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys38, Lys45, Lys282, and Lys413. 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 groups interact with positively charged Lys282 together with Lys45 and/or Lys413. This positions one DIDS isothiocyanate group close to Lys38, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys38 or Lys290 of MCT1 and Lys160 or Lys164 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 Lys38 in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys38 accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp302– and Asp306+, an ion pair already identified as important for transport and located adjacent to Phe360, 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.
|Translated title of the contribution||Studies on the DIDS-binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle|
|Pages (from-to)||20011 - 20021|
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|Early online date||27 May 2009|
|Publication status||Published - 24 Jul 2009|
Bibliographical noteOther: Supplemental material can be found at http://www.jbc.org/cgi/content/full/M109.014217/DC1
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HPC (High Performance Computing) Facility
Sadaf R Alam (Manager), Steven A Chapman (Manager), Polly E Eccleston (Other), Simon H Atack (Other) & D A G Williams (Manager)