12 research outputs found
Membrane Composition Influences the Activity of <i>in Vitro</i> Refolded Human Vitamin K Epoxide Reductase
Human vitamin K epoxide reductase
(hVKOR) is an integral membrane
protein responsible for the maintenance of reduced vitamin K pools,
a prerequisite for the action of γ-glutamyl carboxylase and
hence for hemostasis. Here we describe the recombinant expression
of hVKOR as an insoluble fusion protein in <i>Escherichia coli</i>, followed by purification and chemical cleavage under denaturing
conditions. <i>In vitro</i> renaturation and reconstitution
of purified solubilized hVKOR in phospholipids could be established
to yield active protein. Crucially, the renatured enzyme is inhibited
by the powerful coumarin anticoagulant warfarin, and we demonstrate
that enzyme activity depends on lipid composition. The completely
synthetic system for protein production allows a rational investigation
of the multiple variables in membrane protein folding and paves the
way for the provision of pure, active membrane protein for structural
studies
Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris
The biosynthesis of -terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of -terpinene synthase (TPS). Purified -terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to -terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata
Structure and Dynamics of Meprin β in Complex with a Hydroxamate-Based Inhibitor
The astacin protease Meprin β represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin β inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin β holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors
Crystallization and preliminary X-ray diffraction analysis of transaldolase from Thermoplasma acidophilum
2.7 Å cryo-EM structure of vitrified M. Musculus H-chain apoferritin from a compact 200 keV cryo-microscope
Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution
Outward open conformation of a Major Facilitator Superfamily multidrug/H⁺ antiporter provides insights into switching mechanism
薬剤耐性の原因「薬剤汲み出しタンパク質」の排出メカニズムを解明 --多剤排出トランスポーターMdfAの分子機構-- 京都大学プレスリリース. 2018-10-03.Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H⁺ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters
Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases
Crystal Structures of Glutaminyl Cyclases (QCs) from <i>Drosophila melanogaster</i> Reveal Active Site Conservation between Insect and Mammalian QCs
Glutaminyl cyclases (QCs), which catalyze the formation
of pyroglutamic
acid (pGlu) at the N-terminus of a variety of peptides and proteins,
have attracted particular attention for their potential role in Alzheimer’s
disease. In a transgenic <i>Drosophila melanogaster</i> (<i>Dm</i>) fruit fly model, oral application of the potent competitive
QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified
Aβ. In contrast to mammals such as humans and rodents, there
are at least three <i>Dm</i>QC species, one of which (isoDromeQC)
is localized to mitochondria, whereas DromeQC and an isoDromeQC splice
variant possess signal peptides for secretion. Here we present the
recombinant expression, characterization, and crystal structure determination
of mature DromeQC and isoDromeQC, revealing an overall fold similar
to that of mammalian QCs. In the case of isoDromeQC, the putative
extended substrate binding site might be affected by the proximity
of the N-terminal residues. PBD150 inhibition of DromeQC is roughly
1 order of magnitude weaker than that of the human and murine QCs.
The inhibitor binds to isoDromeQC in a fashion similar to that observed
for human QCs, whereas it adopts alternative binding modes in a DromeQC
variant lacking the conserved cysteines near the active center and
shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing
an explanation for the lower affinity. Our biophysical and structural
data suggest that isoDromeQC and human QC are similar with regard
to functional aspects. The two <i>Dm</i> enzymes represent
a suitable model for further in-depth analysis of the catalytic mechanism
of animal QCs, and isoDromeQC might serve as a model system for the
structure-based design of potential AD therapeutics