Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats

Abstract

Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 Å apart in the crystal structures of GltPh, but Ϸ10 Å apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline) 3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-␤-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.alternating access ͉ conformationally sensitive cross-linking ͉ homology modeling ͉ neurotransmitter ͉ secondary transport

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…A similar concept was used previously to study the electrostatics of ion permeation in ClC channels (46) and to model alternate conformations of secondary transporters (3,47,48). In this way, we predicted that the helices corresponding to A1 and B3 in the opposite repeat, i.e., helices B1 and A3 (Fig.…”

“…At the same time, the fact that the binding sites in BetP are located at sites that are related to one another by the symmetry in the FIRL-fold suggests that considering the inverted-topology repeats may be useful in studies of various aspects of transport (3,47,48).…”

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

“…L-Aspartate as the preferred substrate exhibits the highest association rates and HP2 closure rate (22). The closure of HP2 is followed by substrate translocation via an "elevatorlike" mechanism (15,25), where the transport domain, which includes HP1, HP2, and TM3 and -6 -8, moves relative to the fixed trimerization domain (26) by around 15 Å. The lateral movement of the transport domain at intermediate positions results in the formation of an anion-selective conduction pathway (27) and thus permits prokaryotic (28) and eukaryotic transporters (29,30) to operate as glutamate-gated anion channels.…”

Conserved Asparagine Residue Located in Binding Pocket Controls Cation Selectivity and Substrate Interactions in Neuronal Glutamate Transporter

“…Importantly, many of the amino acid residues of the transporter, inferred to be important in the interaction with sodium (24,25), potassium (6,26), and glutamate (27,28) are facing toward the binding pocket. Recent studies indicate that glutamate translocation occurs by an "elevator-like" mechanism (29,30) where the transport domain, which includes HP1 and HP2 and TMs 3, 6, 7, and 8, moves relative to the fixed trimerization domain (31).…”