Skip to main content

Linking the intrinsic protein dynamics to function in glutamate transporters

Periodic Reporting for period 1 - MEMDYN (Linking the intrinsic protein dynamics to function in glutamate transporters)

Reporting period: 2016-04-01 to 2018-03-31

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. This conformational change provides alternating access of the aspartate binding-site to opposite sides of the membrane, leading to transport when coupled to an ion gradient. Aspartate is bound between two helical hairpins on the periphery of the transport domain that roughly face towards the center of the trimer in OFS. At least one hairpin opens and closes to bind or release aspartate. More recently, structures of human SLC1 members show that they share the GltPh-fold, suggesting a conserved functional mechanism through an elevator movement. While structures provide snapshots of the transport mechanism, it is unclear which parts of the protein determine the rates of the conformational change. 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. It then aims to correlate the rates of elevator movement with local dynamics of structural elements in the GltPh-fold.
To probe sequence variation in a systematic manner, we drew on the observation that the temperature of the optimal enzyme activity correlates strongly with the temperature of the habitats of the originating organisms. Thus, thermophilic enzymes are slow compared to the mesophilic enzymes at ambient temperatures. We developed a bioinformatics approach to identify systematic amino acid changes that might be associated with temperature adaptation across bacterial glutamate transporters. We used this analysis as a guideline to introduce substitutions into GltPh with the aim to augment its transport activity at ambient temperatures without modifying the substrate-binding site. Gain-of-function was assessed by the transport of radiolabeled aspartate into artificial liposomes.
Over 30 single substitution variants were created. Top scorers, which increased transport up to five-fold, were further combined with each other, with functionally neutral substitutions and with previously documented gain-of-function GltPh-variants. This strategy resulted into GltPh-variants that increased the aspartate transport rate up to 25-fold.
A panel of GltPh-variants containing representative transporters across the catalytic range was selected for structure determination and measurement of elevator dynamics. So far, crystal structures at medium and high-resolution of several GltPh-variants did not reveal striking differences with structures of the wild-type protein. Elevator dynamics were measured by distance changes associated with the OFS-to-IFS reorientation of the substrate-binding domains using energy transfer between two site-specific fluorophores as a molecular ruler at the single molecule level by total internal reflection microscopy. These results showed that increased transport rates correlate well with increased elevator dynamics in single substitution variants. However, measurements on multi-substitution variants with the higher transport rates failed to confirm this correlation.
To determine the mechanism of accelerated aspartate transport in multi-substitution variants, we measured substrate affinities and release rates from the IFS. Although substitutions were selected to conserve the substrate binding site, aspartate affinities were lowered in some variants, but these changes did not appear to correlate linearly with increased transport rates, even though aspartate release by IFS-cross-linked GltPh-variants suggested that release was accelerated in multi-substitution variants.
Further details will be available from our publications:

1. Huysmans GHM*, Ciftci D, Wang X, Blanchard SC and Boudker O*. The high-energy transition state of a membrane transporter. *co-corresponding authors
2. Matin TR, Heath GR, Huysmans GHM, Boudker O and Scheuring S. Millisecond dynamics of unlabeled amino acid transporters.
3. Huang Y, Wang X, Lv G, Razavi A, Huysmans GH, Weinstein H, Bracken C, Elizier D and Boudker O. Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog.
4. Ciftci D, Huysmans GH, Wang X, He C, Terry D, Zhou Z, Fitzgerald G, Blanchard SC and Boudker O. Single-Molecule Transport Kinetics of a Glutamate Transporter Homologue Shows Static Disorder.
The current view of membrane protein transport cycles is dominated by snapshots of transporters in different conformations along the cycle, like OFS and IFS for GltPh. To link structure to function, methods are required that can follow individual transporters at work and the structural elements that determine the transport rate are required to be identified.
In this project we have developed a bioinformatics approach to select gain-of-function GltPh-variants and examined these by single molecule microscopy. We correlated gain-of-function properties (in part) with an increased frequency of the elevator movement. Thus, we identified the elements of the GltPh structure that control the elevator rate and provide a glimpse of the rate-limiting transition state structure.
Further studies, aim to provide a structural perspective on these findings by crystallization of additional variants and assessment of changes in local dynamics using a variety of strategies, including hydrogen-deuterium exchange coupled to mass spectrometry.
Overall, these studies provide a framework to identify regions in GltPh that control its functional dynamics. Our approach might prove widely applicable to other membrane protein folds of pharmacological importance and provide new clues for drug design.
GltPh conformational change leading to aspartate transport