Christopher J. Law

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.

Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures.

Sertraline and fluoxetine are selective serotonin re-uptake inhibitors (SSRIs) that are widely prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess halogen atoms at specific positions, which are key determinants for the drugs specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT markedly reduces the transporters affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only nonconserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the proteins HBP.

Rings, ellipses and horseshoes: how purple bacteria harvest solar energy.

This Review summarises the current state of research on the structure and function of light-harvesting apparatus in purple photosynthetic bacteria. Particular emphasis is placed on the major open questions still outstanding in this field in addition to what is already known.

Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT.

Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P(i)) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.

The structure and function of bacterial light-harvesting complexes (Review)

The harvesting of solar radiation by purple photosynthetic bacteria is achieved by circular, integral membrane pigment-protein complexes. There are two main types of light-harvesting complex, termed LH2 and LH1, that function to absorb light energy and to transfer that energy rapidly and efficiently to the photochemical reaction centres where it is trapped. This mini-review describes our present understanding of the structure and function of the purple bacterial light-harvesting complexes.

UPHILL ENERGY TRANSFER IN LH2-CONTAINING PURPLE BACTERIA AT ROOM TEMPERATURE

Uphill energy transfer in the LH2-containing purple bacteria Rhodopseudomonas acidophila, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Chromatium vinosum and Chromatium purpuratum was studied by stationary fluorescence spectroscopy at room temperature upon selective excitation of the B800 pigments of LH2 and the B880 pigments of LH1 at 803 nm and 900 nm, respectively. The resulting fluorescence spectra differed significantly at wavelengths shorter than the fluorescence maximum but agreed at longer wavelengths. The absorption spectra of the species studied were decomposed into five bands at approx. 800, 820, 830, 850 and 880 nm using the shapes of the absorption spectra of the LH1-RC only species Rhodospirillum rubrum and the isolated B800-850 complex from Rps. acidophila strain 10050 as guide spectra. This allowed a quantification of the number of pigments in each pigment group and, consequently, the antenna size of the photosynthetic unit assuming 36 bacteriochlorophyll a molecules in an LH1-RC complex. In most of the LH2-containing purple bacterial strains the number of LH2 rings per LH1-RC was less than the idealized number of eight (Papiz et al., Trends Plant Sci. 1 (1996) 198-206), which was achieved only by C. purpuratum. Uphill energy transfer was assayed by comparing the theoretical fluorescence spectrum obtained from a Boltzmann equilibrium with the measured fluorescence spectrum obtained by 900 nm excitation. The good match of both spectra in all the purple bacteria studied indicates that uphill energy transfer occurs practically up to its thermodynamically maximal possible extent. All strains studied contained a small fraction of either poorly connected or unconnected LH2 complexes as indicated by higher fluorescence yields from the peripheral complexes than predicted by thermal equilibration or kinetic modeling. This impedes generally the quantitative analysis of blue-excited fluorescence spectra.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E.u2009coli may encounter in human gut that MdtM, a single‐component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E.u2009coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single‐component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H+ antiport.

The effect of chemical oxidation on the fluorescence of the LH1 (B880) complex from the purple bacterium Rhodobium marinum.

The effect of chemical oxidation on the absorption and fluorescence emission spectra of the LH1 complex from Rhodobium marinum was investigated. Mild chemical oxidation of the LH1 complex, by addition of 10 mM potassium ferricyanide, caused a 2–3% bleaching of the 880‐nm Qy absorption band. In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the LH1 complex was quenched by about 50%. This result demonstrates that oxidation of very few bacteriochlorophyll (BChl) molecules in the LH1 ring is enough to completely quench its fluorescence.

Structural Basis of Substrate Selectivity in the Glycerol-3-Phosphate: Phosphate Antiporter GlpT

Major facilitators represent the largest superfamily of secondary active transporter proteins and catalyze the transport of an enormous variety of small solute molecules across biological membranes. However, individual superfamily members, although they may be architecturally similar, exhibit strict specificity toward the substrates they transport. The structural basis of this specificity is poorly understood. A member of the major facilitator superfamily is the glycerol-3-phosphate (G3P) transporter (GlpT) from the Escherichia coli inner membrane. GlpT is an antiporter that transports G3P into the cell in exchange for inorganic phosphate (P(i)). By combining large-scale molecular-dynamics simulations, mutagenesis, substrate-binding affinity, and transport activity assays on GlpT, we were able to identify key amino acid residues that confer substrate specificity upon this protein. Our studies suggest that only a few amino acid residues that line the transporter lumen act as specificity determinants. Whereas R45, K80, H165, and, to a lesser extent Y38, Y42, and Y76 contribute to recognition of both free P(i) and the phosphate moiety of G3P, the residues N162, Y266, and Y393 function in recognition of only the glycerol moiety of G3P. It is the latter interactions that give the transporter a higher affinity to G3P over P(i).