RT Journal Article
SR Electronic
T1 Heterogeneous substrate binding in a glutamate transporter homologue
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.07.08.451670
DO 10.1101/2021.07.08.451670
A1 Reddy, Krishna D.
A1 Ciftci, Didar
A1 Scopelliti, Amanda
A1 Boudker, Olga
YR 2021
UL http://biorxiv.org/content/early/2021/07/09/2021.07.08.451670.abstract
AB Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport.Competing Interest StatementThe authors have declared no competing interest.