The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter:sodium symporters (NSS) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild type (WT) and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we utilized distance measurements between spin label pairs to elucidate Na+- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The novel conformational changes reported here present a dynamic picture of the alternating access mechanism of LeuT and NSS that is different to the inferences reached from currently available crystal structures.

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native–like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60-80Å) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuTfold has been captured in outward-facing, occluded, and inwardfacing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na + -coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion-and substrate-dependent conformational equilibria. In contrast to the Na + /leucine transporter LeuT, our results suggest that Na + binding at the conserved second Na + binding site does not change the energetics of the inward-and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.EPR | DEER | Na + -coupled symport | conformational dynamics | transport mechanism

Secondary active transporters couple the uphill translocation of substrates to electrochemical ion gradients. Transporter conformational motion, generically referred to as alternating access, enables a central ligand binding site to change its orientation relative to the membrane. Here we review themes of alternating access and the transduction of ion gradient energy to power this process in the LeuT-fold class of transporters where crystallographic, computational and spectroscopic approaches have converged to yield detailed models of transport cycles. Specifically, we compare findings for the Na+-coupled amino acid transporter LeuT and the Na+-coupled hydantoin transporter Mhp1. Although these studies have illuminated multiple aspects of transporter structures and dynamics, a number of questions remain unresolved that so far hinder understanding transport mechanisms in an energy landscape perspective.

SignificanceTransporters isomerize between conformations to shuttle cargo across membranes, but the mechanism is not understood. Double electron–electron resonance measurements on the sodium-dependent sugar transporter (vSGLT) were used to explore the conformational state of the transporter under specific ligand conditions. Although sugar transport by vSGLT is driven by sodium gradients, vSGLT adopts an inward-open conformation irrespective of the presence of sodium. In the presence of sodium and galactose, the transporter transitions to an occluded conformation. We propose that the cell’s negative membrane potential aids in driving vSGLT toward the outward-facing state to bind sugar and begin the transport cycle. These findings could be applicable to other transporters whereby the inherent cellular membrane potential is integrated into the transport cycle.

A detailed understanding of the functional mechanism of a protein entails the characterization of its energy landscape. Achieving this ambitious goal requires the integration of multiple approaches including determination of high resolution crystal structures, uncovering conformational sampling under distinct biochemical conditions, characterizing the kinetics and thermodynamics of transitions between functional intermediates using spectroscopic techniques, and interpreting and harmonizing the data into novel computational models. With increasing sophistication in solution-based and ensemble-oriented biophysical approaches such as electron paramagnetic resonance (EPR) spectroscopy, atomic resolution structural information can be directly linked to conformational sampling in solution. Here, we detail how recent methodological and technological advances in EPR spectroscopy have contributed to the elucidation of membrane protein mechanisms. Furthermore, we aim to assist investigators interested in pursuing EPR studies by providing an introduction to the technique, a primer on experimental design, and a description of the practical considerations of the method towards generating high quality data.

A hybrid protein structure determination approach combining sparse Electron Paramagnetic Resonance (EPR) distance restraints and Rosetta de novo protein folding has been previously demonstrated to yield high quality models (Alexander et al., 2008). However, widespread application of this methodology to proteins of unknown structures is hindered by the lack of a general strategy to place spin label pairs in the primary sequence. In this work, we report the development of an algorithm that optimally selects spin labeling positions for the purpose of distance measurements by EPR. For the α-helical subdomain of T4 lysozyme (T4L), simulated restraints that maximize sequence separation between the two spin labels while simultaneously ensuring pairwise connectivity of secondary structure elements yielded vastly improved models by Rosetta folding. 50% of all these models have the correct fold compared to only 21% and 8% correctly folded models when randomly placed restraints or no restraints are used, respectively. Moreover, the improvements in model quality require a limited number of optimized restraints, the number of which is determined by the pairwise connectivities of T4L α-helices. The predicted improvement in Rosetta model quality was verified by experimental determination of distances between spin labels pairs selected by the algorithm. Overall, our results reinforce the rationale for the combined use of sparse EPR distance restraints and de novo folding. By alleviating the experimental bottleneck associated with restraint selection, this algorithm sets the stage for extending computational structure determination to larger, traditionally elusive protein topologies of critical structural and biochemical importance.

Excessive consumption of fructose in the Western diet has been associated with metabolic disorders such as type 2 diabetes and obesity. Altered expression and activity of the fructose uniporter GLUT5, a member of the family of GLUT transporters that facilitate the diffusion of monosaccharides across membranes, has been linked to such disorders. Using Saccharomyces cerivisiae as an expression host, and fluorescence-based methods, we identified mammalian GLUT5 orthologues that are suitable for biochemical and structural studies. Here, we present a crystal structure of GLUT5 from Bos Taurus (bovine) in an inward-facing conformation, refined against data extending tõ 3.1 Å resolution. Like GLUT1, GLUT5 shows the typical Major Facilitator Superfamily (MFS) fold, which consists of two 6-TM bundles, and four additional helices that form a soluble domain on the cytoplasmic side of the membrane. The substrate-binding site is highly similar to that observed in the recent crystal structure of human GLUT1, and to those of other GLUT isoforms based on amino acid sequence. However, there are notable differences. In particular, the substrate-binding site of GLUT5 is larger, because the equivalent of a tryptophan residue lining the cavity that contains the substrate-binding site in GLUT1 is an alanine in GLUT5. Furthermore, we have identified a single point mutation that switches the substrate preference of GLUT5 from D-fructose to D-glucose. Overall, our structural and biochemical data provide novel insights into the structure and substrate specificity of GLUT5, a member of the family of the medically relevant GLUT transporters.