Bedaquiline inhibits the yeast and human mitochondrial ATP synthases.

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis.

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily.

The rapid increase of multidrug resistance poses urgent threats to human health. Multidrug transporters prompt multidrug resistance by exporting different therapeutics across cell membranes, often by utilizing the H electrochemical gradient. MdfA from Escherichia coli is a prototypical H -dependent multidrug transporter belonging to the Major Facilitator Superfamily.

Structure of an engineered multidrug transporter MdfA reveals the molecular basis for substrate recognition.

MdfA is a prototypical H-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity. The involvement of specific H-bonds in MdfA-drug interactions and the simplicity of altering the substrate specificity of MdfA contradict the promiscuous nature of multidrug recognition, presenting a baffling conundrum. Here we show the X-ray structures of MdfA variant I239T/G354E in complexes with three electrically different ligands, determined at resolutions up to 2.

High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.

Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded F motor that rotates to drive ATP synthesis in the F subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.

Structure and function of the divalent anion/Na symporter from Vibrio cholerae and a humanized variant.

Integral membrane proteins of the divalent anion/Na symporter (DASS) family translocate dicarboxylate, tricarboxylate or sulphate across cell membranes, typically by utilizing the preexisting Na gradient. The molecular determinants for substrate recognition by DASS remain obscure, largely owing to the absence of any substrate-bound DASS structure. Here we present 2.

Crystallographic study of a MATE transporter presents a difficult case in structure determination with low-resolution, anisotropic data and crystal twinning.

NorM from Neisseria gonorrhoeae (NorM-NG) belongs to the multidrug and toxic compound extrusion (MATE) family of membrane-transport proteins, which can extrude cytotoxic chemicals across cell membranes and confer multidrug resistance. Here, the structure determination of NorM-NG is described, which had been hampered by low resolution (∼ 4 Å), data anisotropy and pseudo-merohedral twinning. The crystal structure was solved using molecular replacement and was corroborated by conducting a difference Fourier analysis.

Structural basis for the blockade of MATE multidrug efflux pumps.

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H(+) or Na(+) electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.

Structural insights into H+-coupled multidrug extrusion by a MATE transporter.

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by coupling the efflux of drugs to the influx of Na(+) or H(+). Known structures of Na(+)-coupled, extracellular-facing MATE transporters from the NorM subfamily revealed 12 membrane-spanning segments related by a quasi-two-fold rotational symmetry and a multidrug-binding cavity situated near the membrane surface. Here we report the crystal structure of an H(+)-coupled MATE transporter from Bacillus halodurans and the DinF subfamily at 3.

Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states.

Oligomycin frames a common drug-binding site in the ATP synthase.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c(10) ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c(10) ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis.

Structure of the c(10) ring of the yeast mitochondrial ATP synthase in the open conformation.

The proton pore of the F(1)F(o) ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state.

Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His(6) tag for Ni-chelate affinity purification.

Crystal structures of mutant forms of the yeast F1 ATPase reveal two modes of uncoupling.

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F(1) ATPase with mgi mutations were crystallized; the structures were solved and analyzed.

Conformational changes in BAK, a pore-forming proapoptotic Bcl-2 family member, upon membrane insertion and direct evidence for the existence of BH3-BH3 contact interface in BAK homo-oligomers.

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein.

Toward effective HIV vaccination: induction of binary epitope reactive antibodies with broad HIV neutralizing activity.

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures.

Asymmetric structure of the yeast F1 ATPase in the absence of bound nucleotides.

The crystal structure of nucleotide-free yeast F(1) ATPase has been determined at a resolution of 3.6 A. The overall structure is very similar to that of the ground state enzyme.

Autoantibody-catalyzed hydrolysis of amyloid beta peptide.

We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenström's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor.

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications.

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.

Regulation through the RNA polymerase secondary channel. Structural and functional variability of the coiled-coil transcription factors.

Gre factors enhance the intrinsic endonucleolytic activity of RNA polymerase to rescue arrested transcription complexes and are thought to confer the high fidelity and processivity of RNA synthesis. The Gre factors insert the extended alpha-helical coiled-coil domains into the RNA polymerase secondary channel to position two invariant acidic residues at the coiled-coil tip near the active site to stabilize the catalytic metal ion. Gfh1, a GreA homolog from Thermus thermophilus, inhibits rather than activates RNA cleavage.

Structure of sperm-specific protein SSP-19 from Caenorhabditis elegans.

Structural data are reported for SSP-19, a sperm-specific protein (SSP) family member from Caenorhabditis elegans. The SSP family [also known as the major sperm protein-like (MSP-like) family] contains proteins with only 107-109 amino acids, compared with 127 amino acids in the major sperm protein (MSP) family. MSP, the most abundant protein in nematode sperm, forms a dynamic actin-like cytoskeleton that provides the framework for the nematode sperm motility.

Expression, purification, crystallization of fragments from the C-terminal region of DFF45/ICAD.

DFF45/ICAD, which forms a heterodimer with DFF40/CAD as its DNase inhibitor and chaperone, plays a key role in nuclei DNA fragmentation in apoptosis. Several fragments from the C-terminal region of DFF45/ICAD have been cloned and expressed in Escherichia coli as His-tagged proteins. After purification to homogeneity, the recombinant proteins of three fragments were crystallized by the hanging-drop vapor-diffusion method.

Purification, nanocrystallization and preliminary X-ray analysis of a C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans.

The C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans was expressed in Escherichia coli and purified to homogeneity. Optimized from the initial nanoscreen, crystals grew to dimensions of 0.25 x 0.

NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.

The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5.