scholarly journals The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state

2006 ◽  
Vol 395 (2) ◽  
pp. 345-353 ◽  
Author(s):  
Stéphanie Ravaud ◽  
Marie-Ange Do Cao ◽  
Marie Jidenko ◽  
Christine Ebel ◽  
Marc Le Maire ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Size Exclusion ◽  
Supramolecular Organization ◽  
Oligomeric State ◽  
Atp Binding Cassette Transporter ◽  
Bound Lipids ◽  
Exclusion Chromatography ◽  
Stokes Radius

BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents. Furthermore, the ATPase activity of BmrA and the quantity of bound lipids and detergent were determined, and the oligomeric state was analysed using SEC (size-exclusion chromatography) and analytical ultracentrifugation. The activity and the quaternary structure of BmrA appeared to be strongly influenced by the type and concentration of the detergent used. SEC data showed that BmrA could be purified in a functional form in 0.05 and 0.01% DDM (n-dodecyl-β-D-maltoside) and was homogeneous and monodisperse with an Rs (Stokes radius) of 5.6 nm that is compatible with a dimer structure. Sedimentation-velocity and equilibrium experiments unequivocally supported that BmrA purified in DDM is a dimer and excluded the presence of other oligomeric states. These observations, which are discussed in relation to results obtained in proteoliposomes, also constitute an important first step towards crystallographic studies of BmrA structure.

scholarly journals Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

2015 ◽  
Vol 71 (4) ◽  
pp. 986-995 ◽  
Author(s):  
C. M. D. Swarbrick ◽  
M. A. Perugini ◽  
N. Cowieson ◽  
J. K. Forwood
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Size Exclusion ◽  
X Ray Scattering ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Enzyme Families

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on theEscherichia colistructure.Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of theY. pestisTesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

scholarly journals Structure of the Prx6-subfamily 1-Cys peroxiredoxin from Sulfolobus islandicus

2019 ◽  
Vol 75 (6) ◽  
pp. 428-434 ◽  
Author(s):  
Sander Stroobants ◽  
Inge Van Molle ◽  
Queen Saidi ◽  
Karl Jonckheere ◽  
Dominique Maes ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Quaternary Structure ◽  
Size Exclusion ◽  
Molecular Replacement ◽  
Oligomeric State ◽  
Data Set ◽  
Cell Parameters ◽  
Aeropyrum Pernix ◽  
Sulfolobus Islandicus ◽  
Exclusion Chromatography

Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Å resolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = β = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

scholarly journals The Oligomeric State of the Plasma Membrane H+-ATPase from Kluyveromyces lactis

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 958 ◽  
Author(s):  
Yadira Ruiz-Granados ◽  
Valentín De La Cruz-Torres ◽  
José Sampedro
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Quaternary Structure ◽  
Kluyveromyces Lactis ◽  
Size Exclusion ◽  
Oligomeric State ◽  
Aggregation State ◽  
Activated State ◽  
Exclusion Chromatography ◽  
Native Electrophoresis

The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Coiled-coil deformations in crystal structures: themeasles virusphosphoprotein multimerization domain as an illustrative example

2014 ◽  
Vol 70 (6) ◽  
pp. 1589-1603 ◽  
Author(s):  
David Blocquel ◽  
Johnny Habchi ◽  
Eric Durand ◽  
Marion Sevajol ◽  
François Ferron ◽  
...  
Keyword(s):  
Measles Virus ◽  
Quaternary Structure ◽  
Dynamic Equilibrium ◽  
Crystal Packing ◽  
Coiled Coil ◽  
Terminal Region ◽  
Size Exclusion ◽  
Structural Deformation ◽  
Structural Comparison ◽  
Exclusion Chromatography

The structures of two constructs of themeasles virus(MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communieet al.(2013),J. Virol.87, 7166–7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.

scholarly journals Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Morgane Agez ◽  
Elodie Desuzinges Mandon ◽  
Thomas Iwema ◽  
Reto Gianotti ◽  
Florian Limani ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Analytical Ultracentrifugation ◽  
B Cell Lymphoma ◽  
Integral Membrane Protein ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Extraction And Purification ◽  
Exclusion Chromatography ◽  
Specificity Of Binding

Abstract CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

scholarly journals Homodimerization of calpain 3 penta-EF-hand domain

2005 ◽  
Vol 388 (2) ◽  
pp. 585-591 ◽  
Author(s):  
Ravikiran RAVULAPALLI ◽  
Beatriz GARCIA DIAZ ◽  
Robert L. CAMPBELL ◽  
Peter L. DAVIES
Keyword(s):  
Skeletal Muscle ◽  
Molecular Modelling ◽  
Analytical Ultracentrifugation ◽  
Domain Size ◽  
Small Subunit ◽  
Size Exclusion ◽  
Relative Molecular Mass ◽  
Calpain 3 ◽  
Ef Hand ◽  
Exclusion Chromatography

Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass Mr 80000) and small (Mr 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution. Molecular modelling suggests that there would be no barriers to the dimerization of the whole enzyme through the PEF domains. This orientation would place the catalytic centres at opposite ends of the dimer.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection

2012 ◽  
Vol 444 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Magali Maitre ◽  
Stéphanie Weidmann ◽  
Aurélie Rieu ◽  
Daphna Fenel ◽  
Guy Schoehn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Dynamic Equilibrium ◽  
Building Blocks ◽  
Oenococcus Oeni ◽  
Size Exclusion ◽  
Acidic Ph

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5–9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.

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