Symmetry in the Structure of the Glutamate Transporter GltPh Suggests Conformation of an Alternate State

Abstract

Glutamate transporters regulate synaptic concentrations of L-glutamate to prevent excitotoxicity in nerve cells. Current crystal structures of GltPh, an archeal homologue of the Glutamate transporters, have an extracellular-facing binding site. The alternating access theory implies that a cytoplasm-facing state also exists. In order to model this state, we have identified two distinct sets of inverted-topology repeats, and used these repeats to model an inward-facing conformation of the protein. Specifically, we modeled the sequence of each repeat on the structure of its partner. In this model, a portion of the protein containing two transmembrane helices (TM7 and 8) and two helical hairpins (HP1 and HP2) is displaced relative to the crystal structure so that the binding site is exposed to the cytoplasm. In order to validate our model, pairs of cysteines were introduced into the neuronal glutamate transporter EAAC1 at positions that were greater than 27 Angstroms apart in the outward-facing crystal structure, but closer to 10 Angstroms apart in our model. Transport in these mutants was activated by pretreatment with the reducing agent dithithreitol. Once treated with the oxidizing agent copper(II)(1,10-phenantroline)3, however, activity ceased. Importantly, this inhibition was potentiated under conditions expected to promote the inward-facing conformation. This suggests that during the transport cycle these cysteines come within the range necessary to crosslink, as predicted by our inverted-topology repeat model of the cytoplasm-facing state. Previously, an alternative conformational state of the LeuT transporter was also modeled using inverted-topology repeats, suggesting that inverted-topology repeats may provide a general and elegant solution to the requirement for two symmetry-related states in a single protein.