Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

Tassis, Konstantinos; Vietrov, Ruslan; Koning, Matthijs de; Boer, Marijn de; Gouridis, Giorgos; Cordes, Thorben

doi:10.1101/2020.08.07.241463

Cited by 4 publications

(7 citation statements)

References 76 publications

(134 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both crystallography and single-molecule Förster-resonance energy transfer (smFRET) experiments on the single SBD1 and SBD2 showed that substrate binding was linked to a conformational change of the corresponding SBD from an open apo conformation to a closed liganded conformation [18][19][20][21]. The results also implied an induced-fit-type ligand-binding mechanism, where conformational dynamics are induced by ligand-SBD interactions similar as later also demonstrated for other SBPs [22][23][24][25][26]. Additionally, it was shown that the opening of the SBDs and ligand release was the rate-limiting step in the transport cycle and that the closed conformation triggers ATP-hydrolysis and transport [18].…”

Section: Introductionsupporting

confidence: 58%

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Ploetz

Schuurman-Wolters

Zijlstra

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.HIGHLIGHTSResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactisConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2No cooperative effects are seen for different ligands for SBDs in the tandemProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously

show abstract

Section: Introductionsupporting

confidence: 58%

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Ploetz

Schuurman-Wolters

Zijlstra

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…These assays include antibodies that recognize structural epitopes, [89] thermal unfolding assays of the protein in the presence and absence of ligands, [89] binding assays via bilayer interferometry [89] or Surface Plasmon Resonance [90] or other biophysical and biochemical techniques. [ 90 , 91 , 92 , 93 , 94 , 95 , 96 ] The impact of mutations on function can be monitored by ATPase assays[ 79 , 90 , 97 , 98 , 99 , 100 , 101 ] or transport assays. [ 78 , 90 , 93 , 94 , 96 , 97 , 100 , 102 , 103 , 104 , 105 , 106 , 107 , 108 ] In transport assays, the protein is either overexpressed in a cell or incorporated into liposomes and the accumulation of fluorescent[ 78 , 97 , 105 ] or radiolabeled substrate[ 90 , 93 , 94 , 96 , 100 , 102 , 103 , 106 , 107 ,…”

Section: Challenges Of Studying Transporters With Smfret and How To Overcome Themmentioning

confidence: 99%

“…[ 90 , 91 , 92 , 93 , 94 , 95 , 96 ] The impact of mutations on function can be monitored by ATPase assays[ 79 , 90 , 97 , 98 , 99 , 100 , 101 ] or transport assays. [ 78 , 90 , 93 , 94 , 96 , 97 , 100 , 102 , 103 , 104 , 105 , 106 , 107 , 108 ] In transport assays, the protein is either overexpressed in a cell or incorporated into liposomes and the accumulation of fluorescent[ 78 , 97 , 105 ] or radiolabeled substrate[ 90 , 93 , 94 , 96 , 100 , 102 , 103 , 106 , 107 , 108 ] in the compartment (cell or vesicle) is measured. Good examples for such careful controls are LmrP transporter variants whose activities were checked with the fluorescent ligand Hoechst, [105] or variants of the Glt Ph transporter in liposomes where the uptake of the radioactive substrate [ 3 H]‐Asp was used to confirm activity.…”

Section: Challenges Of Studying Transporters With Smfret and How To Overcome Themmentioning

confidence: 99%

“…SmFRET studies were carried out on prokaryotic (MsbA, McjD, BtuCD from E. coli and OpuA from Lactococcus lactis ) and eukaryotic (PgP from Mus musculus and MRP1 from Bos Taurus ) ABC transporters. [ 90 , 97 , 98 , 99 , 100 , 101 ] Out of these, OpuA and BtuCD are characterized as importers whereas all others are exporters.…”

Section: Recent Smfret Studies On Transportersmentioning

confidence: 99%

See 1 more Smart Citation

Single‐Molecule FRET of Membrane Transport Proteins

et al. 2021

View full text Add to dashboard Cite

Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane-embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high-resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state-of-the-art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design.

show abstract

“…This highlights why ABC transporter structural biology has to be accompanied by complementary methods, may they be spectroscopy‐based, mechanistic, or cell‐based. Additionally, advances in single molecule techniques and theoretical approaches show great potential to help investigate these aspects of ABC transporter mechanisms [8–12]. A major conclusion is that structural information should always be interpreted taking into consideration all available biochemical and genetic studies, as well as the technical limitations of in vitro membrane‐mimicking environments, as opposed to the native membrane of a living cell [13–17].…”

Section: Understanding Abc Transporter Function From Structurementioning

confidence: 99%