MEMDYN · Linking the intrinsic protein dynamics to function in glutamate transporters

Linking the intrinsic protein dynamics to function in glutamate transporters

The transport of molecules across biological membranes is essential in all forms of life. To achieve this, biological membranes contain permanently embedded proteins, e.g. transporters, that facilitate the exchange of nutrients and ions with the environment. Glutamate is the primary chemical used for transmission in the brain and is involved in many brain functions, as well as playing a role in the pathogenesis of neurological disorders. Historically, atomic detail of transporter function has come mostly from the study of bacterial transporter homologues. Structural understanding of glutamate transporters (solute carrier family 1 (SLC1)) mostly comes from high-resolution crystal structures of the aspartate transporter from the archaeal hyperthermophilic bacterium Pyrococcus horikoshii, GltPh. These structures show that GltPh forms trimers in which three substrate-binding domains move as independent elevators across the membrane from a physiologically outward facing state (OFS) to an inward facing state (IFS) against a static trimerization scaffold. Aspartate binds between two pseudo symmetric helical hairpins (HPs) that alternately interact with the scaffold, of which the most extracellular one (HP2) opens and closes to bind and release substrate. The OFS to IFS transition provides alternating access of the aspartate binding-site to opposite sides of the membrane, leading to transport when coupled to an ion gradient. The transition is fast in the absence of substrate and rate-limiting to the transport cycle when aspartate is bound. While structures provide snapshots of the transport mechanism, it is unclear which parts of the protein determine the rates of the conformational change and, thus, which movement limits transport. This understanding is crucial if we are to come to a detailed understanding of SLC1 function and want to increase our ability to design drug compounds that modulate activity in disease. This project aims to identify which areas of the GltPh-fold determine the rate of alternating access by comparing sequence variation between bacterial homologues in the SLC1 family. The distribution and dynamic properties of gain-of-function mutations indicate that the high-energy transition-state for the OFS to IFS conformational change is close to the IFS. We conclude that disengaging HP1 from the scaffold in OFS is easy and that GltPh makes many attempts to form a stable interaction between HP2 and the scaffold to reach a stable IFS.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз