Da-Neng Wang

Ins and Outs of Major Facilitator Superfamily Antiporters

The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.

LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

Tricyclic antidepressants exert their pharmacological effect—inhibiting the reuptake of serotonin, norepinephrine, and dopamine—by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.

Three-dimensional map of the dimeric membrane domain of the human erythrocyte anion exchanger, Band 3.

The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein consisting of a 43 kDa N‐terminal cytosolic domain that binds the cytoskeleton, haemoglobin and glycolytic enzymes and a 52 kDa C‐terminal membrane domain that mediates anion transport. Electron microscopy and three‐dimensional image reconstruction of negatively stained two‐dimensional crystals of the dimeric membrane domain revealed a U‐shaped structure with dimensions of 60 × 110 A, and a thickness of 80 A. The structure is open on the top and at the sides, with the monomers in close contact at the base. The basal domain is 40 A thick and probably spans the lipid bilayer. The upper part of the dimer consists of two elongated protrusions measuring 25 × 80 A in projection, with a thickness of 40 A. The protrusions form the sides of a canyon, enclosing a wide space that narrows down and converges into a depression at the centre of the dimer on the top of the basal domain. This depression may represent the opening to a transport channel located at the dimer interface. Based on the available protein‐chemical data, the two protrusions face the cytosolic side of the membrane and they appear to be dynamic.

Electron microscopic analysis of KvAP voltage-dependent K+ channels in an open conformation.

Voltage-dependent ion channels serve as field-effect transistors by opening a gate in response to membrane voltage changes. The gates response to voltage is mediated by voltage sensors, which are arginine-containing structures that must move with respect to the membrane electric field. We have analysed by electron microscopy a voltage-dependent K+ channel from Aeropyrum pernix (KvAP). Fab fragments were attached to ‘voltage sensor paddles’ and identified in the electron microscopy map at 10.5 Å resolution. The extracellular surface location of the Fab fragments in the map is consistent with the membrane-depolarized, open conformation of the channel in electrophysiological experiments. Comparison of the map with a crystal structure demonstrates that the voltage sensor paddles are ‘up’ (that is, near the channels extracellular surface) and situated at the protein–lipid interface. This finding supports the hypothesis that in response to changes in voltage the sensors move at the protein–lipid interface rather than in a gating pore surrounded by protein.

High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.

Large two-dimensional crystals of the light-harvesting chlorophyll a/b-protein complex (LHC-II) from the photosynthetic membrane of pea chloroplasts were grown by a new method from detergent solution. The structure of these crystals was examined by electron crystallography, using three different media to preserve high-resolution detail: vitrified water, glucose and tannin. The crystals diffracted electrons to at least 3.2 A resolution in all three media. R-factors between the three data sets of electron diffraction amplitudes ranged from 6.4% to 14.3%. Fourier difference maps were generated and compared to a projection map of the complex at 3.4 A resolution. No significant differences were found, proving that all three media preserved the native structure of LHC-II at high resolution. The probability of recording high-quality electron diffraction patterns with tannin was 90%. With glucose and water this probability was lower by a factor of 10 to 20, suggesting that tannin may be preferable as a preserving medium for sensitive biological specimens.

Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane.

The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two‐dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six‐fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two‐fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 × 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 × 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.

Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: A member of the major facilitator superfamily

Here we report the successful three‐dimensional crystallization of GlpT, the glycerol‐3‐phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane α‐helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol‐3‐phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size‐exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better‐ordered crystals. These crystals exhibited order to 3.7 Å resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 Å resolution and were suitable for structure determination by X‐ray crystallography.

Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter

In human cells, cytosolic citrate is a chief precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the energy balance of the cell by activating the fatty-acid-synthesis pathway while downregulating both the glycolysis and fatty-acid β-oxidation pathways. The rate of fatty-acid synthesis in liver and adipose cells, the two main tissue types for such synthesis, correlates directly with the concentration of citrate in the cytosol, with the cytosolic citrate concentration partially depending on direct import across the plasma membrane through the Na+-dependent citrate transporter (NaCT). Mutations of the homologous fly gene (Indy; I’m not dead yet) result in reduced fat storage through calorie restriction. More recently, Nact (also known as Slc13a5)-knockout mice have been found to have increased hepatic mitochondrial biogenesis, higher lipid oxidation and energy expenditure, and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance. To understand the transport mechanism of NaCT and INDY proteins, here we report the 3.2 Å crystal structure of a bacterial INDY homologue. One citrate molecule and one sodium ion are bound per protein, and their binding sites are defined by conserved amino acid motifs, forming the structural basis for understanding the specificity of the transporter. Comparison of the structures of the two symmetrical halves of the transporter suggests conformational changes that propel substrate translocation.

THE STRUCTURE OF CRYSTALLINE BACTERIAL SURFACE LAYERS

THREE-DIMENSIONAL STRUCTURES OF S-LAYERS 1. Classification of S-layers According to Symmetry 2. Hexagonal S-layers (a) Sulfolobus acidocaldarius (b) Thermoproteus tenax (c) Synechocystis sp. CLII (d) Eubacterium sp. AHN 990 (e) Clostridium thermohydrosulfuricum (f) Deinococcus radiodurans (g) Aquaspirillum serpens (h) Acetooenium kivui (i) Bacteroides buccae 3. Tetragonal S-layers (a) Bacillus sphae~icus (b) Sporosarcina ureae (c) Clostridium aceticum (d) Clostridium thermosaccharolyticum (e) Eubacterium sp. ES4C (f) Aeromonas hydrofila (g) Azotobacter vinelandi (h) Desulfurococcus mobilis Oblique S-layers (a) Methanospirillum hungatei 4.

DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli

Overexpression of CorA, the major magnesium transporter from bacterial inner membrane, in Escherichia coli resulted in the synthesis of 60mg of protein per liter of culture, most of which however was in the form of inclusion bodies. The levels of inclusion body formation were reduced by lowering the cell culture temperature. To dissect CorA inclusion body formation and the folding process involved, we co-expressed the protein with various chaperones and other folding modulators. Expression of DnaK/DnaJ (Hsp70) prevented inclusion bodies from forming and resulted in the integration of more CorA into the membrane. GroEL/GroES (Hsp60/Hsp10) were less effective at reducing CorA inclusion body formation. Co-expression with either Ffh/4.5S-RNA, the signal recognition particle, or SecA, the ATPase that drives protein insertion into the membrane, had little effect on CorA folding. These results indicate: (1) that CorA inclusion bodies form immediately after synthesis at 37 degrees C, (2) that CorA solubility in the cytosol can be increased by co-expressing a chaperone system, (3) membrane targeting is probably not a rate-limiting factor, and (4) that membrane insertion becomes a limitation only when large amounts of soluble CorA are present in the cytosol. These co-expression systems can be used for producing other membrane proteins in large quantities.