Membrane co-transport proteins that utilize a 5-helix inverted repeat motif have recently emerged as one of the largest structural class of secondary active transporters1,2. However, despite many structural advances there is no clear evidence as to how ion and substrate transport are coupled. Here, we report a comprehensive study of the Sodium-Galactose Transporter from Vibrio parahaemolyticus (vSGLT) consisting of molecular dynamics simulations, biochemical characterization, and a new crystal structure of the inward-open conformation at 2.7 Å resolution. Our data show that sodium exit causes a reorientation of transmembrane helix 1 (TM1) opening an inner gate required for substrate exit, while also triggering minor rigid body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose to readily enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium binding sites, the unwound portion of TM1, and the substrate binding site that is essential in the coupling of co-transport.

The Na ؉ -Ca 2؉ exchanger plays a central role in cardiac contractility by maintaining Ca 2؉ homeostasis. Two Ca 2؉ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca 2؉ binding to these regulatory domains activates the transport of Ca 2؉ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca 2؉ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca 2؉ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca 2؉ ions in primary and secondary Ca 2؉ sites. In the absence of Ca 2؉ , Lys 585 stabilizes the structure by coordinating two acidic residues (Asp 552 and Glu 648 ), one from each of the Ca 2؉ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca 2؉ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca 2؉ site completely remove Ca 2؉ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na ؉ -Ca 2؉ exchanger.calcium binding ͉ calcium regulation

The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.bioenergetics | membrane protein crystal structure | MTS reagents | sugar binding | affinity labeling T he Major Facilitator Superfamily (MFS) is one of the largest and most diverse families of membrane transporters with members from Archae to Homo sapiens that utilize uniport, symport, or antiport mechanisms to transport a broad range of substrates across membranes (1). Most members of the family have 12 to 14 transmembrane segments and catalyze transport by an alternating access mechanism (1-3). The lactose permease of Escherichia coli (LacY) is the most intensively studied representative of the MFS and embodies a paradigm for understanding general transport mechanisms throughout the superfamily.LacY catalyzes a symport reaction-the coupled translocation of a H þ and a galactopyranoside (galactoside∕H þ symport). Because translocation is obligatorily coupled, sugar accumulation against a concentration gradient is achieved by using the free energy released from the downhill movement of H þ with the electrochemical H þ gradient (Δμ H þ ; interior negative and/or alkaline). Conversely, downhill sugar translocation by LacY drives uphill translocation of H þ with the generation of Δμ H þ , the polarity of which depends on the direction of the sugar concentration gradient (4).Several crystal structures of LacY have been resolved, from both a conformationally restricted mutant C154G (5, 6) and the wild-type protein (7), all of which exhibit the same overall architecture. The protein is composed of 12 transmembrane helices organized in two pseudosymmetrical six α-helical bundles surrounding a large hydrophilic cavity open solely to the cytoplasm representing an inward-facing conformation. The sugar-binding site and the residues involved in H þ translocation are near the apex of the cavity, approximately in the middle of the molecule. For the most part, residues involved in sugar recognition are confined to the N-terminal bundle, while those important for H þ translocation are located in the C-terminal bundle. Systematic mutagenesis of each residue in LacY has identified less than 10 irreplaceable residues absolutely required for lactose∕H þ symport. E126 (helix IV) and R144 (helix V) are essential for substrate r...

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

P2X receptors (P2X1-P2X7) are cell surface cation channels that transition from closed to open states upon binding ATP. They represent a physiologically and medically important (1), as well as a structurally distinct family of ligand-gated ion channels that are quite different to Cys-loop and ionotropic glutamate receptors (2-5). P2X receptors are found in many species from several phyla, implying important roles in diverse life forms (6, 7). A key goal is to understand how P2X receptors operate at the chemical level.The first P2X receptor genes were cloned in 1994 (8, 9) leading to important progress over the last decade in understanding how P2X receptors function (10, 11) using biophysical and biochemical approaches. P2X subunits are thus known to possess intracellular N and C termini and two transmembrane (TM) 2 segments separated by a large extracellular loop (12-14). The trimeric architecture of P2X receptors is also well established based on subunit concatemers, blue-native PAGE, and atomic force microscopy experiments (15-18). It is also well established that the P2X pore is lined by TM2 (19 -22), with TM1 making little contribution to ion flow (23, 24). Moreover, careful studies of permeation and selectivity have suggested that the same area of TM2 is responsible for both ion selection and channel opening (25-31). Mutational approaches have shed light on the extracellular domain of P2X receptors, including how the ATP binding pocket may form (32), how the 10 conserved cysteines contribute to the fold of the protein (33, 34), and how cations such as Zn 2ϩ profoundly affect receptor function (35)(36)(37)(38). Finally, meticulous analysis of voltage-jump relaxations for ATP-evoked currents combined with mutagenesis has suggested that TM2 may display hinge-like flexibility (39, 40), and recently the P2X4 receptor open pore has been imaged with fast scanning atomic force microscopy (41). Most of these past studies were conducted on rat P2X2 (rP2X2) receptors and have provided a basis to explore P2X receptor pores and the gating process.In 2009, the field was immeasurably advanced by the landmark achievement and report of a crystal structure of the zebrafish (zf) P2X4.1 receptor in the closed state (42). The P2X receptor topology was similar to that of acid-sensing ion channels (42, 43), a possibility that had been raised by earlier modeling studies (44). However, it is important to note that the extracellular domains of P2X and acid-sensing ion channels are distinct with little sequence or structural homology, whereas the pore domains share similar architectures (43). The availability of direct structural information for P2X receptors substantiated most of the aforementioned mutational studies and for the first time provided a framework to understand their atomic origins (5). Of most relevance here, it directly revealed that the P2X pore was indeed formed by TM2 ␣-helices arranged around a 3-fold molecular axis of symmetry. These were arranged ϳ45°to the membrane normal and crossed about halfway along their l...

All eukaryotic cells require efficient trafficking of metabolites between the mitochondria and the rest of the cell. This exchange is carried out by the dominant protein in the outer mitochondrial membrane (OMM), the Voltage Dependent Anion Channel (VDAC), which serves as the primary pathway for the exchange of ions and metabolites between the cytoplasm and the intermembrane space of the mitochondria. Additionally, VDAC provides a scaffold for the binding of modulator proteins to the mitochondria and has been implicated in mitochondria-dependent cell death. We recently determined the structure of the murine VDAC1 (mVDAC1) at 2.3Å resolution crystallized in a native-like bilayer environment. The high-resolution structure provided concise structural details about the voltage-sensing N-terminal domain and catalyzed new hypotheses regarding the gating mechanisms for metabolites and ions that transit the OMM. In this study, the crystal packing of mVDAC1 is analyzed revealing a strong antiparallel dimer that further assemble as hexamers mimicking the native oligomeric packing observed in EM and AFM images of the OMM. Oligomerization has been shown to be important for VDAC regulation and function, and mVDAC1 crystal packing in a lipidic medium reveals insights on how oligomerization is accomplished using protein-protein and protein-lipid interactions. Furthermore, orientation of VDAC in the OMM remains uncertain due to inconsistencies in antibody labeling studies. The physiological implications of a novel antiparallel arrangement are addressed that may clarify these conflicting biochemical data.

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