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Treatment of cells expressing rMate1 with p‐chloromercuribenzene sulfonate, an organic mercurial chemical, significantly reduced uptake of TEA. This inhibition of transport could be rescued by dithiothreitol suggesting that reduced sulfhydryl groups are important for the activity of rMate1 [17]. Subsequent site‐directed mutagenesis identified key residues [66]. One histidine (rMate1: His‐385, hMATE1: His‐386, hMATE2‐K: His‐382) and two cysteine residues (rMate1: Cys‐62 and Cys‐126, hMATE1: Cys‐63 and Cys‐127, hMATE2‐K: Cys‐59 and Cys‐123) were essential for transport activity. The impaired function of mutants in these residues was not due to improper trafficking or reduced expression as all localized to the plasma membrane to similar degrees as wild‐type proteins [66]. Interestingly, unlabeled TEA was able to protect against the transport inhibition achieved by the sulfhydryl reagent PCMBS suggesting that rMate1 substrates interact directly with sulfhydryl‐containing Cys‐62 and Cys‐126 [66]. By comparison, unlabeled TEA had no effect on the ability of the histidine residue modifier DEPC to block rMate1 activity in vitro.

There has also been interest in elucidating the role of negatively charged glutamates in the recognition of substrates that are largely cationic in nature. Mutation of the glutamate residue at position 273 to a glycine reduced hMATE1 function without altering its insertion into the plasma membrane [5]. Notably, this glutamate residue is conserved across rMate1, mMate1, and hMATE2‐K. Substitution of glutamate at position 273 with alanine resulted in the absence of hMATE1 protein, whereas amino acid changes at the remaining glutamates differentially altered activity and affinity for cimetidine and TEA. For example, aspartate mutants at each glutamate residue decreased overall transporter activity; however, Glu273Asp increased affinity for cimetidine but reduced affinity for TEA [67]. Purified MATE1 protein containing glutamine at position 273 (rather than glutamate) further support this residue as a binding site for organic cations [48]. Within the hMATE1 homology model based on the N or M structure, the four glutamate residues localize to the hydrophilic cleft, which would likely represent the path for substrate translocation [63]. Whether all four of these glutamates are directly involved in substrate binding or contribute to overall secondary structure stability is unclear but nonetheless impact hMATE1 transporter activity.

Using the crystal structure of AtDTX14, it has been proposed that eukaryotic MATE proteins rely upon conformation changes in the 7th transmembrane domain. The 7th transmembrane domain is predicted to undergo protonation at a conserved acidic residue in the C‐lobe that leads to electrostatic repulsion and a bent conformation. Bending of this domain is anticipated to collapse the cavity of the C‐lobe leading to hydrogen‐binding and release of substrate. While in the straight conformation, the proton is absent and the 7th transmembrane domain would be capable of binding positively charged substrates [65]. As the 7th transmembrane domain moves from the straight to the bent confirmation, the proteins are expected to undergo “rocking” between the inward‐open and outward‐open configurations.