5 research outputs found
Exploring conformational equilibria of a heterodimeric ABC transporter
No full textABC exporters pump substrates across the membrane by coupling ATP-driven
movements of nucleotide binding domains (NBDs) to the transmembrane domains
(TMDs), which switch between inward- and outward-facing (IF, OF) orientations.
DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga
maritima, which contains a non-canonical ATP binding site, revealed that in
the presence of nucleotides the transporter exists in an IF/OF equilibrium.
While ATP binding was sufficient to partially populate the OF state,
nucleotide trapping in the pre- or post-hydrolytic state was required for a
pronounced conformational shift. At physiologically high temperatures and in
the absence of nucleotides, the NBDs disengage asymmetrically while the
conformation of the TMDs remains unchanged. Nucleotide binding at the
degenerate ATP site prevents complete NBD separation, a molecular feature
differentiating heterodimeric from homodimeric ABC exporters. Our data suggest
hydrolysis-independent closure of the NBD dimer, which is further stabilized
as the consensus site nucleotide is committed to hydrolysis
Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5
No full textLegionella pneumophila can cause Legionnaires’ disease and replicates intracellularly in a
distinct Legionella-containing vacuole (LCV). LCV formation is a complex process that
involves a plethora of type IV-secreted effector proteins. The effector RidL binds the Vps29
retromer subunit, blocks retrograde vesicle trafficking, and promotes intracellular bacterial
replication. Here, we reveal that the 29-kDa N-terminal domain of RidL (RidL2–281) adopts a
“foot-like” fold comprising a protruding β-hairpin at its “heel”. The deletion of the β-hairpin,
the exchange to Glu of Ile170 in the β-hairpin, or Leu152 in Vps29 abolishes the interaction in
eukaryotic cells and in vitro. RidL2–281 or RidL displace the Rab7 GTPase-activating protein
(GAP) TBC1D5 from the retromer and LCVs, respectively, and TBC1D5 promotes the
intracellular growth of L. pneumophila. Thus, the hydrophobic β-hairpin of RidL is critical for
binding of the L. pneumophila effector to the Vps29 retromer subunit and displacement of the
regulator TBC1D5
CLIC2-RyR1 Interaction and Structural Characterization by Cryo-electron Microscopy
No full textChloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [(3)H] ryanodine binding, Ca(2+) efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [(3)H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the a parent maximal binding capacity; (2) CLIC2 reduced the maximal Ca(2+) efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels. (C) 2009 Elsevier Ltd. All rights reserved.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000264941400006&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biochemistry & Molecular BiologySCI(E)29ARTICLE2320-33438
Synthetic single domain antibodies for the conformational trapping of membrane proteins
No full textMechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins
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Publisher Correction: Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus (Nature Microbiology, (2019), 10.1038/s41564-019-0376-y)
No full textIn the version of this Article originally published, the affiliation for author Catherine Linard was incorrectly stated as '6Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK'. The correct affiliation is '9Spatial Epidemiology Lab (SpELL), Universite Libre de Bruxelles, Brussels, Belgium'. The affiliation for author Hongjie Yu was also incorrectly stated as '11Department of Statistics, Harvard University, Cambridge, MA, USA'. The correct affiliation is '15School of Health, Fudan University, Key Laboratory of Public Health Safety, Ministry of Education, Shanghai, China'. This has now been amended in all versions of the Article
