The ancient and ubiquitous major facilitator superfamily (MFS) represents the largest secondary transporter family and plays a crucial role in a multitude of physiological processes. MFS proteins ...transport a broad spectrum of ions and solutes across membranes via facilitated diffusion, symport, or antiport. In recent years, remarkable advances in understanding the structural biology of the MFS transporters have been made. This article reviews the history, classification, and general features of the MFS proteins; summarizes recent structural progress with a focus on the sugar porter family transporters exemplified by GLUT1; and discusses the molecular mechanisms of substrate binding, alternating access, and cotransport coupling.
The major facilitator superfamily (MFS) is one of the largest groups of secondary active transporters conserved from bacteria to humans. MFS proteins selectively transport a wide spectrum of ...substrates across biomembranes and play a pivotal role in multiple physiological processes. Despite intense investigation, only seven MFS proteins from six subfamilies have been structurally elucidated. These structures were captured in distinct states during a transport cycle involving alternating access to binding sites from either side of the membrane. This review discusses recent progress in MFS structure analysis and focuses on the molecular basis for substrate binding, co-transport coupling, and alternating access.
The cellular uptake of glucose is an essential physiological process, and movement of glucose across biological membranes requires specialized transporters. The major facilitator superfamily glucose ...transporters GLUTs, encoded by the SLC2A genes, have been a paradigm for functional, mechanistic, and structural understanding of solute transport in the past century. This review starts with a glimpse into the structural biology of membrane proteins and particularly membrane transport proteins, enumerating the landmark structures in the past 25years. The recent breakthrough in the structural elucidation of GLUTs is then elaborated following a brief overview of the research history of these archetypal transporters, their functional specificity, and physiological and pathophysiological significances. Structures of GLUT1, GLUT3, and GLUT5 in distinct transport and/or ligand-binding states reveal detailed mechanisms of the alternating access transport cycle and substrate recognition, and thus illuminate a path by which structure-based drug design may be applied to help discover novel therapeutics against several debilitating human diseases associated with GLUT malfunction and/or misregulation.
Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. Three families of secondary glucose transporters have been ...identified in human, including the major facilitator superfamily glucose facilitators GLUTs, the sodium‐driven glucose symporters SGLTs, and the recently identified SWEETs. Structures of representative members or their prokaryotic homologs of all three families were obtained. This review focuses on the recent advances in the structural elucidation of the glucose transporters and the mechanistic insights derived from these structures, including the molecular basis for substrate recognition, alternating access, and stoichiometric coupling of co‐transport.
Animal toxins that modulate the activity of voltage-gated sodium (Na
) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and ...saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Na
channel Na
PaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSD
and the pore of Na
PaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na
access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Na
channel drugs.
1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or ...agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.
High‐resolution cryo‐EM structures of nanodisc‐embedded Cav1.1 in complex with antagonists Levamlodipine, (R)‐(+)‐Bay K8644, and dual agonist/antagonist (S)‐(−)‐Bay K8644 reveal the molecular basis of the allosteric modulation of the voltage‐gated Ca2+ channel Cav1.1 by dihydropyridine (DHP) drugs. Advanced structural understanding of the stereo‐selective mode of action of DHP will facilitate drug discovery targeting Cav channels.
Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron ...cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the threedimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
Targeting sodium channelsVoltage-gated sodium (Nav) channels have been implicated in cardiac and neurological disorders. There are many subtypes of these channels, making it challenging to develop ...specific therapeutics. A core α subunit is sufficient for voltage sensing and ion conductance, but function is modulated by β subunits and by natural toxins that can either act as pore blockers or gating modifiers (see the Perspective by Chowdhury and Chanda). Shen et al. present the structures of Nav1.7 in complex with both β1 and β2 subunits and with animal toxins. Pan et al. present the structure of Nav1.2 bound to β2 and a toxic peptide, the µ-conotoxin KIIIA. The structure shows why KIIIA is specific for Nav1.2. These and other recently determined Nav structures provide a framework for targeted drug development.Science, this issue p. 1303, p. 1309; see also p. 1278Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo–electron microscopy structures of the human Nav1.7-β1-β2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSDII), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSDIV. The structures may represent an inactivated state with all four VSDs “up” and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.
The Hedgehog (Hh) pathway involved in development and regeneration is activated by the extracellular binding of Hh to the membrane receptor Patched (Ptch). We report the cryo-EM structures of human ...Ptch1 alone and in complex with the N-terminal domain of human Sonic hedgehog (ShhN) at resolutions of 3.9 Å and 3.6 Å, respectively. Ptch1 comprises two interacting extracellular domains ECD1 and ECD2 and twelve transmembrane segments (TMs), with TMs 2-6 constituting the sterol-sensing domain (SSD). Two steroid-shaped densities are resolved in both structures, one enclosed by ECD1/2, and the other on the membrane-facing cavity of SSD. Structure-guided mutational analysis shows that interaction between ShhN and Ptch1 is steroid-dependent. The structure of a steroid binding-deficient Ptch1 mutant displays pronounced conformational rearrangements.
The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to ...GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2 Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters.
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