André Schiefner

Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism

The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli.

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-π interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.

The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance.

Antimicrobial resistance of human pathogenic bacteria is an emerging problem for global public health. This resistance is often associated with the overproduction of membrane transport proteins that are capable to pump chemotherapeutics, antibiotics, detergents, dyes and organic solvents out of the cell. In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude a large variety of cytotoxic substances from the cell membrane directly into the medium bypassing the periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump in charge of the efflux of multiple antibiotics, dyes, bile salts and detergents. The trimeric outer membrane factor (OMF) TolC forms a beta-barrel pore in the outer membrane and exhibits a long periplasmic alpha-helical conduit. The periplasmic membrane fusion protein (MFP) AcrA serves as a linker between TolC and the trimeric resistance nodulation cell division (RND) pump AcrB, located in the inner membrane acting as a proton/drug antiporter. The newly elucidated asymmetric structure of trimeric AcrB reveals three different monomer conformations representing consecutive states in a transport cycle. The monomers show tunnels with occlusions at different sites leading from the lateral side through the periplasmic porter (pore) domains towards the funnel of the trimer and TolC. The structural changes create a hydrophobic pocket in one monomer, which is not present in the other two monomers. Minocyclin and doxorubicin, both AcrB substrates, specifically bind to this pocket substantiating its role as drug binding pocket. The energy transduction from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (Asp407/Asp408/Lys940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel transport model merges Jardetzkys alternate access pump mechanism with the rotating site catalysis of F(1)F(0) ATPase and suggests a working hypothesis for the transport mechanism of RND transporters in general.

The Crystal Structure of Mlc, a Global Regulator of Sugar Metabolism in Escherichia coli

Mlc from Escherichia coli is a transcriptional repressor controlling the expression of a number of genes encoding enzymes of the phosphotransferase system (PTS), including ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters. In addition, Mlc controls the transcription of malT, the gene of the global activator of the mal regulon. The inactivation of Mlc as a repressor is mediated by binding to an actively transporting PtsG (EIICBGlc). Here we report the crystal structure of Mlc at 2.7 Å resolution representing the first described structure of an ROK (repressors, open reading frames, and kinases) family protein. Mlc forms stable dimers thus explaining its binding affinity to palindromic operator sites. The N-terminal helix-turn-helix domain of Mlc is stabilized by the amphipathic C-terminal helix implicated earlier in EIICBGlc binding. Furthermore, the structure revealed a metal-binding site within the cysteine-rich ROK consensus motif that coordinates a structurally important zinc ion. A strongly reduced repressor activity was observed when two of the zinc-coordinating cysteine residues were exchanged against serine or alanine, demonstrating the role of zinc in Mlc-mediated repressor function. The structures of a putative fructokinase from Bacillus subtilis, the glucokinase from Escherichia coli, and a glucomannokinase from Arthrobacter sp. showed high structural homology to the ROK family part of Mlc.

The menagerie of human lipocalins: a natural protein scaffold for molecular recognition of physiological compounds.

While immunoglobulins are well-known for their characteristic ability to bind macromolecular antigens (i.e., as antibodies during an immune response), the lipocalins constitute a family of proteins whose role is the complexation of small molecules for various physiological processes. In fact, a number of low-molecular-weight substances in multicellular organisms show poor solubility, are prone to chemical decomposition, or play a pathophysiological role and thus require specific binding proteins for transport through body fluids, storage, or sequestration. In many cases, lipocalins are involved in such tasks. Lipocalins are small, usually monomeric proteins with 150-180 residues and diameters of approximately 40 Å, adopting a compact fold that is dominated by a central eight-stranded up-and-down β-barrel. At the amino-terminal end, this core is flanked by a coiled polypeptide segment, while its carboxy-terminal end is followed by an α-helix that leans against the β-barrel as well as an amino acid stretch in a more-or-less extended conformation, which finally is fixed by a disulfide bond. Within the β-barrel, the antiparallel strands (designated A to H) are arranged in a (+1)7 topology and wind around a central axis in a right-handed manner such that part of strand A is hydrogen-bonded to strand H again. Whereas the lower region of the β-barrel is closed by short loops and densely packed hydrophobic side chains, including many aromatic residues, the upper end is usually open to solvent. There, four long loops, each connecting one pair of β-strands, together form the entrance to a cup-shaped cavity. Depending on the individual structure of a lipocalin, and especially on the lengths and amino acid sequences of its four loops, this pocket can accommodate chemical ligands of various sizes and shapes, including lipids, steroids, and other chemical hormones as well as secondary metabolites such as vitamins, cofactors, or odorants. While lipocalins are ubiquitous in all higher organisms, physiologically important members of this family have long been known in the human body, for example with the plasma retinol-binding protein that serves for the transport of vitamin A. This prototypic human lipocalin was the first for which a crystal structure was solved. Notably, several other lipocalins were discovered and assigned to this protein class before the term itself became familiar, which explains their diverse names in the scientific literature. To date, up to 15 distinct members of the lipocalin family have been characterized in humans, and during the last two decades the three-dimensional structures of a dozen major subtypes have been elucidated. This Account presents a comprehensive overview of the human lipocalins, revealing common structural principles but also deviations that explain individual functional features. Taking advantage of modern methods for combinatorial protein design, lipocalins have also been employed as scaffolds for the construction of artifical binding proteins with novel ligand specificities, so-called Anticalins, hence opening perspectives as a new class of biopharmaceuticals for medical therapy.

Crystallographic analysis of AcrB

A His‐tagged derivative of the multidrug efflux pump AcrB could be crystallized in three different space groups (R3, R32 and P321). Experimental MAD‐phasing maps from R32 AcrBHis crystals were obtained to a resolution of 3.5 Å. Datasets of native and substrate soaked AcrBHis crystals were collected at the Swiss Light Source X06SA beamline up to a resolution of 2.7 Å and refinement of these data provided good quality electron density maps, which allowed us to complement the published AcrB structure (PDB code 1iwg). Introduction of amino acids 860–865 and 868 lacking in the 1iwg structure and deletion of a highly disordered region (amino acids 669–678) improved R free and average B factors in the 2.7 Å model. We could not identify significant densities indicating specific antibiotic binding sites in the AcrB R32 space group datasets under the soaking conditions tested.

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

Background: Extracellular matrix and plasma forms of fibronectin show fibrillar and compact conformations, respectively. Results: X-ray analysis of the FnIII7B89 fragment reveals a rotation between domains 7 and 8 upon insertion of B and, unexpectedly, homodimerization. Conclusion: ED-B in oncofetal Fn appears to stabilize oligomerization. Significance: Together with alternating disulfide bridges at the C-terminal tail, the homodimerization suggests a model for macromolecular fibril formation. The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment FnIII7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the FnIII10 domain, its RGD loop as well as the adhesion synergy region in FnIII9–10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.

Crystallization and preliminary X-ray analysis of Mlc from Escherichia coli

Mlc is a prokaryotic transcriptional repressor controlling the expression of a number of genes encoding enzymes of the Escherichia coli phosphotransferase system (PTS), ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters, as well as malT, the gene of the global activator of the mal regulon. The mlc gene has been cloned into a pQE vector and recombinant protein with the point mutation R52H was expressed and purified as the selenomethionine-labelled derivative. Crystallization of SeMet-Mlc R52H was carried out using the vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 235.95, b = 74.71, c = 154.95 A, beta = 129.15 degrees, and diffract to 2.9 A resolution.

Crystallization and preliminary X-ray analysis of Aes, an acetyl-esterase from Escherichia coli.

Aes belongs to the family of hormone-sensitive lipases and has acetyl-esterase activity. It is also known to control maltose uptake through interaction with MalT, the central regulator of the Escherichia coli maltose system. Aes was crystallized as an N-terminally His(6)-tagged protein both in the native form and with selenomethionine substitution. Crystals grew in both cases in space group R32 to dimensions of about 0.2 x 0.15 x 0.05 mm (native His(6)-Aes) and about 0.5 x 0.3 x 0.1 mm (SeMet-His(6)-Aes). A native data set has been obtained at 2.4 A resolution; the selenomethionine-substituted Aes crystals diffracted to 3.0 A resolution.

Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis

Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.