Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.
J Biol Chem
; 299(2): 102805, 2023 02.
Article
in En
| MEDLINE
| ID: mdl-36529287
ABSTRACT
EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protonsone cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Antiporters
/
Catalytic Domain
/
Escherichia coli Proteins
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Escherichia coli
/
Static Electricity
/
Glutamates
Language:
En
Journal:
J Biol Chem
Year:
2023
Document type:
Article
Affiliation country:
United States