1 research outputs found
Closure of the Cytoplasmic Gate Formed by TM5 and TM11 during Transport in the Oxalate/Formate Exchanger from <i>Oxalobacter formigenes</i>
OxlT, the oxalate/formate exchanger
of Oxalobacter
formigenes, is a member of the major facilitator superfamily
of transporters. In the present work, substrate (oxalate) was found
to enhance the reactivity of the cysteine mutant S336C on the cytoplasmic
end of helix 11 to methanethiosulfonate ethyl carboxylate. In addition,
S336C is found to spontaneously cross-link to S143C in TM5 in either
native or reconstituted membranes under conditions that support transport.
Continuous wave EPR measurements are consistent with this result and
indicate that positions 143 and 336 are in close proximity in the
presence of substrate. These two residues are localized within helix
interacting GxxxG-like motifs (G<sub>140</sub>LASG<sub>144</sub> and
S<sub>336</sub>DIFG<sub>340</sub>) at the cytoplasmic poles of TM5
and TM11. Pulse EPR measurements were used to determine distances
and distance distributions across the cytoplasmic or periplasmic ends
of OxlT and were compared with the predictions of an inside-open homology
model. The data indicate that a significant population of transporter
is in an outside-open configuration in the presence of substrate;
however, each end of the transporter exhibits significant conformational
heterogeneity, where both inside-open and outside-open configurations
are present. These data indicate that TM5 and TM11, which form part
of the transport pathway, transiently close during transport and that
there is a conformational equilibrium between inside-open and outside-open
states of OxlT in the presence of substrate