Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1.

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 LeuT-fold has been captured in outward-facing, occluded, and inward-facing 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.

Algorithm for selection of optimized EPR distance restraints for de novo protein structure determination.

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. 54% 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, 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.

Alternating access mechanisms of LeuT-fold transporters: trailblazing towards the promised energy landscapes.

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.

Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy.

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 toward generating high quality data.

Conformational dynamics of ligand-dependent alternating access in LeuT.

The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type 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 used 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 conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy.

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 Å-80 Å) 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.