Studies of structural determinants of substrate binding in the Creatine Transporter (CreaT, SLC6A8) using molecular models.

Creatine is a crucial metabolite that plays a fundamental role in ATP homeostasis in tissues with high-energy demands. The creatine transporter (CreaT, SLC6A8) belongs to the solute carrier 6 (SLC6) transporters family, and more particularly to the GABA transporters (GATs) subfamily. Understanding the molecular determinants of specificity within the SLC6 transporters in general, and the GATs in particular is very challenging due to the high similarity of these proteins. In the study presented here, our efforts focused on finding key structural features involved in binding selectivity for CreaT using structure-based computational methods. Due to the lack of three-dimensional structures of SLC6A8, our approach was based on the realization of two reliable homology models of CreaT using the structures of two templates, i.e. the human serotonin transporter (hSERT) and the prokaryotic leucine transporter (LeuT). Our models reveal that an optimal complementarity between the shape of the binding site and the size of the ligands is necessary for transport. These findings provide a framework for a deeper understanding of substrate selectivity of the SLC6 family and other LeuT fold transporters.

A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport.

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.

Assessing the ability of sequence-based methods to provide functional insight within membrane integral proteins: a case study analyzing the neurotransmitter/Na+ symporter family.

BACKGROUND: Efforts to predict functional sites from globular proteins is increasingly common; however, the most successful of these methods generally require structural insight. Unfortunately, despite several recent technological advances, structural coverage of membrane integral proteins continues to be sparse. ConSequently, sequence-based methods represent an important alternative to illuminate functional roles. In this report, we critically examine the ability of several computational methods to provide functional insight within two specific areas. First, can phylogenomic methods accurately describe the functional diversity across a membrane integral protein family? And second, can sequence-based strategies accurately predict key functional sites? Due to the presence of a recently solved structure and a vast amount of experimental mutagenesis data, the neurotransmitter/Na+ symporter (NSS) family is an ideal model system to assess the quality of our predictions. RESULTS: The raw NSS sequence dataset contains 181 sequences, which have been aligned by various methods. The resultant phylogenetic trees always contain six major subfamilies are consistent with the functional diversity across the family. Moreover, in well-represented subfamilies, phylogenetic clustering recapitulates several nuanced functional distinctions. Functional sites are predicted using six different methods (phylogenetic motifs, two methods that identify subfamily-specific positions, and three different conservation scores). A canonical set of 34 functional sites identified by Yamashita et al. within the recently solved LeuTAa structure is used to assess the quality of the predictions, most of which are predicted by the bioinformatic methods. Remarkably, the importance of these sites is largely confirmed by experimental mutagenesis. Furthermore, the collective set of functional site predictions qualitatively clusters along the proposed transport pathway, further demonstrating their utility. Interestingly, the various prediction schemes provide results that are predominantly orthogonal to each other. However, when the methods do provide overlapping results, specificity is shown to increase dramatically (e.g., sites predicted by any three methods have both accuracy and coverage greater than 50%). CONCLUSION: The results presented herein clearly establish the viability of sequence-based bioinformatic strategies to provide functional insight within the NSS family. As such, we expect similar bioinformatic investigations will streamline functional investigations within membrane integral families in the absence of structure.