Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment

Du, Dijun; Neuberger, Arthur; Orr, Mona W.; Newman, Catherine E.; Hsu, Pin-Chia; Samsudin, Firdaus; Harris, Andrzej; Ramos, Leana M.; Debela, Mekdes; Khalid, Syma; Storz, Gisela; Luisi, Ben F.

doi:10.1016/j.str.2020.03.013

Cited by 49 publications

(58 citation statements)

References 75 publications

(96 reference statements)

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“…Comparing the re ned structure, especially the chain C of the AcrB trimer, in C 8 -C 0 -50 to the previously published structures in SMA (6baj) 41 and saposin (6sgu) 50 using Chimera showed an RMSD of 0.7 and 1.5 Å respectively, re ecting their close similarity. Comparison of the maps (Figure S3) or overlay of the SMA and CyclAPol structures (Fig.…”

Section: Comparison To Acrb In Other Amphipathic Environmentsmentioning

confidence: 53%

“…Importantly, the CyclAPols are still compatible with high-resolution cryoEM studies with the resultant 3.2 Å resolution structure of AcrB obtained in CyclAPol C 8 -C 0 -50 being in line with the highest reported resolutions obtained with classical APols. Further this clearly indicates no detriment is observed for cryo-EM as this represents the joint highest resolution AcrB cryoEM structure 41,50 . We noted a signi cant improvement in resolution compared to our in-house AcrB-SMA cryoEM reconstructions, the highest resolution of which is ~ 4.0 A 42 and for which the data acquisition setup and data processing pipelines were comparable.…”

Section: Discussionmentioning

confidence: 73%

“…The local resolution is lower at exterior helices, where density for the side-chains could not be unambiguously resolved. The previously derived EM structure of AcrB in SMA 41 was used as a starting point for model building and re nement, with the resultant model being highly similar to previously published AcrB structures 41,42,50 . The structure is asymmetric and exhibits a clear cavity at the interior of the trimer, which after model tting was devoid of any signi cant density that could be assigned to lipids (Fig.…”

Section: Single Particle Cryoem Of Acrb In Cyclapol C 8 -C 0 -50mentioning

confidence: 86%

“…AcrB is an ideal model protein for such studies as it has been so widely characterised with high-resolution cryoEM structures being determined in SMA (3.2 Å), 41 saposin (3.2 Å) 50 and most recently in liposomes (3.9 Å) 52 . Studies in saposin 50 and liposomes 52 involve reconstitution subsequent to detergent puri cation. However, AcrB within liposomes also does not appear to show closely associated internal lipids, unlike structures determined in both saposin and SMA.…”

Section: Discussionmentioning

confidence: 99%

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Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Higgins

Flynn

Marconnet

et al. 2021

Preprint

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Membrane proteins are essential for cellular growth and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a significant advantage. Here we use a novel type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful detergent-free approach for the study of membrane proteins allowing native extraction and high-resolution structure determination by cryoEM.

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Section: Comparison To Acrb In Other Amphipathic Environmentsmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 73%

Section: Single Particle Cryoem Of Acrb In Cyclapol C 8 -C 0 -50mentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Higgins

Flynn

Marconnet

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Unlike MSP, however, there is only a single protein construct used to reconstitute integral membrane proteins of various sizes (Frauenfeld et al 2016 ), with saposin A monomers forming a lipid nanoparticle of appropriate size for the membrane protein of interest. To date, the system has seen successful application in membrane protein cryo-EM studies (Du et al 2020 ). Using saposin A, a thermostabilised β 1 AR construct was reconstituted with dimyristoyl PC lipids (Chien et al 2017 ).…”

Section: Saposin a Nanoparticlesmentioning

confidence: 99%

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

Lavington

Watts

2020

Biophys Rev

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G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.

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Microproteins—Discovery, structure, and function

Mohsen,

Martel,

Slavoff

2023

Proteomics

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Advances in proteogenomic technologies have revealed hundreds to thousands of translated small open reading frames (sORFs) that encode microproteins in genomes across evolutionary space. While many microproteins have now been shown to play critical roles in biology and human disease, a majority of recently identified microproteins have little or no experimental evidence regarding their functionality. Computational tools have some limitations for analysis of short, poorly conserved microprotein sequences, so additional approaches are needed to determine the role of each member of this recently discovered polypeptide class. A currently underexplored avenue in the study of microproteins is structure prediction and determination, which delivers a depth of functional information. In this review, we provide a brief overview of microprotein discovery methods, then examine examples of microprotein structures (and, conversely, intrinsic disorder) that have been experimentally determined using crystallography, cryo‐electron microscopy, and NMR, which provide insight into their molecular functions and mechanisms. Additionally, we discuss examples of predicted microprotein structures that have provided insight or context regarding their function. Analysis of microprotein structure at the angstrom level, and confirmation of predicted structures, therefore, has potential to identify translated microproteins that are of biological importance and to provide molecular mechanism for their in vivo roles.

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Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment

Abstract: Highlights d Structure of an RND transporter with an allosteric modulator in a membrane environment d Cooperation of lipid and small protein in allosterically modulating transport activity

Cited by 49 publications

References 75 publications

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

Microproteins—Discovery, structure, and function

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