Abstract
Solute transport, cell signaling, vesicle fusion, and many other important cellular functions are mediated by membrane proteins, which are coded for by approximately 30% of the genome and represent 60% of current drug targets. One important class of membrane proteins is the secondary active transporters, which mediate small molecule transport by harnessing the free energy stored in ion gradients. While there have been an increasing number of crystal structures of in recent years, secondary active transporters remain underrepresented structurally. Although crystal structures provide vital structural information, they do not always provide structures of biologically relevant conformations that are sampled during the transport cycle.
The solute carrier 1 family (SLC1) of secondary active transporters are responsible for neutral and charged amino acid transport, and defects in these transporters have been implicated in many central nervous systems and mental disorders such as Alzheimer’s disease, epilepsy, obsessive/compulsive disorder, and schizophrenia. Gltph is a sodium dependent aspartate transporter that is structurally homologous to the SLC1 family and the only member that has been crystallized. The series of crystal structures in substrate free, substrate bound, and inhibitor bound states have provided an outline of global conformational exchange events during the transport cycle. However, small-scale conformational exchange events that must mediate transport have remained uncharacterized. Furthermore, the impact of the lipid environment on conformational exchange has not been described.
In the present work, Gltph conformational exchange events are described under conditions that support transport. We propose a novel conformation for hairpin loop 1 (HP1) that mediates substrate release intracellularly based on CW-EPR spectra. We also show that the protective osmolyte, sucrose, modulates conformational exchange differently in detergent micelles than in lipid bilayers based on DEER distance distributions, which has implications for the conformations seen in crystal structures. Furthermore, we show that the choice of spin label and lipids used in reconstitution modulate conformational populations. Finally, we propose, based on power saturation depth measurements, that the majority of Gltph is buried in the lipid bilayer, in contrast to current structural models. The work presented here provides explanations for previous contrasting research and provides details on the transport cycle under biologically relevant conditions.
