Structural Information from Spin-Labelled Membrane-Bound Proteins

2013 ◽  
pp. 205-248 ◽  
Author(s):  
Johann P. Klare ◽  
Heinz-Jürgen Steinhoff
Keyword(s):  
Structural Information ◽  
Membrane Bound

scholarly journals Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

2019 ◽  
Vol 75 (4) ◽  
pp. 270-277
Author(s):  
Florian Bleffert ◽  
Joachim Granzin ◽  
Holger Gohlke ◽  
Renu Batra-Safferling ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Structural Information ◽  
Sequence Similarity ◽  
Expression Host ◽  
Parkinson’S Diseases ◽  
Attractive Therapeutic Target ◽  
Membrane Bound ◽  
Bacterial Homolog ◽  
Hydrolyzing Enzymes ◽  
Hydrolysis Of ◽  
Suitable Expression

The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein fromPseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence ofn-octyl β-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified fromEscherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space groupI4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.

scholarly journals Cryo-electron tomography reveals structural insights into the membrane binding and remodeling activity of dynamin-like EHDs

2021 ◽  
Author(s):  
Arthur A. Melo ◽  
Thiemo Sprink ◽  
Jeffrey K. Noel ◽  
Elena Vázquez Sarandeses ◽  
Chris van Hoorn ◽  
...  
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Spontaneous Curvature ◽  
Membrane Remodeling ◽  
Closed Conformation ◽  
Membrane Surfaces ◽  
Cryo Electron Tomography ◽  
Membrane Bound ◽  
Subtomogram Averaging ◽  
Structural Insights

AbstractDynamin-related Eps15-homology domain containing proteins (EHDs) oligomerize on membrane surfaces into filaments leading to membrane remodeling. EHD crystal structures in an open and a closed conformation were previously reported, but structural information on the membrane-bound EHD oligomeric structure has remained enigmatic. Consequently, mechanistic insight into EHD-mediated membrane remodeling is lacking. Here, by using cryo-electron tomography and subtomogram averaging, we determined the structure of an EHD4 filament on a tubular membrane template at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests the AMPPNP-bound filament has a spontaneous curvature of approximately 1/70 nm-1. Combining the available structural and functional data, we propose a model of EHD-mediated membrane remodeling.

scholarly journals Exploring the free-energy landscape of GPCR activation

2018 ◽  
Vol 115 (41) ◽  
pp. 10327-10332 ◽  
Author(s):  
Raphael Alhadeff ◽  
Igor Vorobyov ◽  
Han Wool Yoon ◽  
Arieh Warshel
Keyword(s):  
Free Energy ◽  
Structural Information ◽  
Large Fraction ◽  
Energy Landscape ◽  
Point Of View ◽  
Coarse Grained ◽  
Free Energy Landscape ◽  
Activation Process ◽  
Gpcr Activation ◽  
Membrane Bound

G-protein–coupled receptors (GPCRs) are a large group of membrane-bound receptor proteins that are involved in a plethora of diverse processes (e.g., vision, hormone response). In mammals, and particularly in humans, GPCRs are involved in many signal transduction pathways and, as such, are heavily studied for their immense pharmaceutical potential. Indeed, a large fraction of drugs target various GPCRs, and drug-development is often aimed at GPCRs. Therefore, understanding the activation of GPCRs is a challenge of major importance both from fundamental and practical considerations. And yet, despite the remarkable progress in structural understanding, we still do not have a translation of the structural information to an energy-based picture. Here we use coarse-grained (CG) modeling to chart the free-energy landscape of the activation process of the β-2 adrenergic receptor (β2AR) as a representative GPCR. The landscape provides the needed tool for analyzing the processes that lead to activation of the receptor upon binding of the ligand (adrenaline) while limiting constitutive activation. Our results pave the way to better understand the biological mechanisms of action of the β2AR and GPCRs, from a physical chemistry point of view rather than simply by observing the receptor’s behavior physiologically.

scholarly journals Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

2006 ◽  
Vol 281 (43) ◽  
pp. 32089-32094 ◽  
Author(s):  
Pierre Tremouilhac ◽  
Erik Strandberg ◽  
Parvesh Wadhwani ◽  
Anne S. Ulrich
Keyword(s):  
Antimicrobial Peptide ◽  
Structural Information ◽  
Transmembrane Helix ◽  
Bound State ◽  
Bacterial Membranes ◽  
H Nmr ◽  
Lipid Ratio ◽  
First Case ◽  
Membrane Bound ◽  
T State

The antimicrobial activity of amphipathic α-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state 2H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125° was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This 2H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d3 substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158° is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an α-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

Three-Dimensional Structure of Cobalt-Tetrammine-ATP Induced Membrane Crystals of Na,K-ATPase

1990 ◽  
Vol 48 (1) ◽  
pp. 110-111
Author(s):  
Elisabeth Skriver ◽  
Hans Hebert ◽  
Urban Kavéus ◽  
Arvid B. Maunsbach
Keyword(s):  
Image Processing ◽  
Sodium Pump ◽  
Structural Information ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Membrane Fragment ◽  
Outer Medulla ◽  
Membrane Bound ◽  
Tilt Series

2-D membrane crystals of renal Na,K-ATPase have been induced to obtain structural information about the sodium pump. Crystalline arrays of the p21 and the p1 type formed during incubation in media containing vanadate and magnesium have been analyzed by image processing and the 3-D structures determined. We report here a new 3-D structure reconstructed from membrane crystal with p4 symmetry that were formed during incubation with cobalt-tetrammine-ATP (Co(NH3)4ATP) which is an MgATP analogue complex that binds to the enzyme and stabilizes it in an E2 conformational state.Membrane-bound Na,K-ATPase purified from renal outer medulla was incubated in 0.5 mM Co(NH3)4ATP and 0.5 mM CoCl2 for one hour at 37°C and then at 4°C for 28 days. Tilt series of crystalline membrane fragment negatively stained with 1% uranyl acetate were recorded in a Jeol 100CX electron microscope at angels from −60° to +60° with increments of 6°. Electron micrographs of the tilt series were digitized into 512 × 512 pixel squares using a EIKONIX 1412 scanner and image processing performed with the "EM" program system.

scholarly journals Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1

2015 ◽  
Vol 112 (3) ◽  
pp. 755-760 ◽  
Author(s):  
Michael J. Capper ◽  
Paul M. O’Neill ◽  
Nicholas Fisher ◽  
Richard W. Strange ◽  
Darren Moss ◽  
...  
Keyword(s):  
Structural Information ◽  
Phase 1 ◽  
Macromolecular Complex ◽  
Future Design ◽  
Antimalarial Agents ◽  
Potential Binding ◽  
In Silico Modeling ◽  
Membrane Bound ◽  
Drug Resistant Strains

Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Qo site (one of two potential binding sites within cytochrome bc1) using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Qo site but bind at the Qi site. The discovery that these compounds bind at the Qi site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Qi also explains the ability of this class to overcome parasite Qo-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.

scholarly journals Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase

1995 ◽  
Vol 308 (2) ◽  
pp. 673-681 ◽  
Author(s):  
G Scotland ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Membrane Fraction ◽  
Structural Information ◽  
Subcellular Fractionation ◽  
Translation System ◽  
Membrane Association ◽  
Cytosol Fraction ◽  
Terminal Domain ◽  
Ionic Detergent ◽  
Membrane Bound

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat ‘dunce-like’ phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5′-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.

scholarly journals Regulation of Bacterial Drug Export Systems

2002 ◽  
Vol 66 (4) ◽  
pp. 671-701 ◽  
Author(s):  
Steve Grkovic ◽  
Melissa H. Brown ◽  
Ronald A. Skurray
Keyword(s):  
Regulatory Networks ◽  
Structural Information ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
E Coli ◽  
Therapeutic Drugs ◽  
Regulatory Systems ◽  
Ligand Interactions ◽  
Regulatory Mutations ◽  
Membrane Bound

SUMMARY The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.

scholarly journals Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

2020 ◽  
pp. jbc.RA120.016297
Author(s):  
Gautier Bailleul ◽  
Callum Robert Nicoll ◽  
Maria Laura Mascotti ◽  
Andrea Mattevi ◽  
Marco W. Fraaije
Keyword(s):  
Structural Model ◽  
Catalytic Properties ◽  
Structural Information ◽  
Transmembrane Helix ◽  
Protein Sequences ◽  
Ancestral Reconstruction ◽  
Binding Domains ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Related Compounds

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral sequence recon¬struction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as AncFMO1 and AncFMO4, respectively. AncFMO1, sharing 89.5 % sequence identity with human FMO1, was successfully expressed as functional enzyme. It displayed typical FMO activity as demonstrated by oxygenating benzydamine, tamoxifen and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the FAD and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

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