Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

Reading, Eamonn; Hall, Zoe; Martens, Chloé; Haghighi, Tabasom; Findlay, Heather E.; Ahdash, Zainab; Booth, Paula J.

doi:10.1002/anie.201709657

Cited by 83 publications

(72 citation statements)

References 21 publications

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“…HDX-MS has emerged as a non-invasive and highly sensitive method for interrogating the conformational dynamics of membrane proteins and their complexes [15][16][17][18] . HDX-MS measures the exchange of backbone amide hydrogen to deuterium at peptide-level resolution [19][20][21] .…”

Section: Mainmentioning

confidence: 99%

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HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Ahdash

Pyle

Allen

et al. 2019

Preprint

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The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

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Section: Mainmentioning

confidence: 99%

“…For example, the analysis of the structural consequences of protein-ligand/drug interactions, and nucleotide dependent protein dynamics; ATP/GTP versus ADP/GDP 23,24 . Moreover, our group and others have developed and applied HDX-MS to study the conformational diversity of challenging membrane protein systems 15,17,18 .…”

Section: Mainmentioning

confidence: 99%

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Ahdash

Pyle

Allen

et al. 2019

Preprint

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“…Monitoring the temporal differences in HDX (ΔHDX) of the GlpG membrane protein within the two different native lipid systems revealed regions that were conformationally sensitive to alterations to their native lipid environment. Parts of this Figure was previously published by Wiley‐VCH by Reading et al …”

Section: Basic But Not Simplistic: Native Nanodiscsmentioning

confidence: 99%

“…Recently, HDX‐MS has been employed to study membrane proteins within native nanodiscs . Reading and colleagues successfully analyzed the conformational dynamics of the Escherichia coli rhomboid protease, GlpG, within native nanodiscs (Figure ).…”

Section: Basic But Not Simplistic: Native Nanodiscsmentioning

confidence: 99%

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments

Reading

2018

Chemistry A European J

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Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted systems which lack the native lipid membrane and cellular environments. However, there has been progress in the use and adaptations of these methods for probing membrane proteins within increasingly more native contexts. In this Concept article the use and utility of structural mass spectrometry techniques for studying membrane proteins within native environments are highlighted.

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“…Having determined the structures of the neddylated and deneddylated CSN-CRL2 complexes, we set off to characterise the local dynamics using HDX-MS. HDX-MS provides peptide-level information on the dynamics of proteins through monitoring the exchange events of amide hydrogens for bulk deuterium in the surrounding solution environment [26][27][28][29][30][31]32 . Here, we performed a set of two differential HDX-MS experiments to determine the effect of: a) CRL2~N8 binding to CSN, denoted as Δ(CSN-CRL2~N8 -CSN), and b) CRL2 binding to CSN, denoted as Δ(CSN WT -CRL2 -CSN WT ) ( Fig.…”

Section: Hdx-ms Reveals a Stepwise Mechanism Of Csn Activationmentioning

confidence: 99%

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

Faull

Lau

Martens

et al. 2018

Preprint

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MAINCullin-RING Ligases (CRLs) are modular, multi-subunit complexes that constitute a major class of ubiquitin E3 ligases 1,2 . CRLs coordinate the ubiquitination of substrates as either a signal for degradation via the 26S proteasome, or to alter the function of the target protein 2,3 . The CRL2 E3 ligase consists of a Cullin-2 (CUL2) scaffold in association with a catalytic RING-box protein (RBX1), with the substrate adaptors Elongin B (ELOB) and C (ELOC) at its N-terminal 4 . When associated with the von Hippel-Lindau (VHL) tumour suppressor substrate receptor, the CRL2 complex is the primary regulator of the Hypoxia Inducible Factor 1-α (HIF-1α) transcription factor 5,6 . Mutations in the interface between VHL, ELOB and ELOC can deactivate CRL2leading to an accumulation of HIF-1α, which can in turn drive tumorigenesis through the over-activation of oncogenes 7 . Moreover, CRL2 has recently been identified as a potential target for small molecular inhibitors and PROTACs -a new class of cancer drugs that promote degradation of tumorigenic gene products 8-10 . These fascinating systems have been described in detail by a number of excellent reviews 1,2,3 .Activation of CRL2, in common with other members of the CRL family, involves a cascade of E1, E2 and E3 enzymes, which conjugate the ubiquitin-like protein NEDD8 (N8) to residue K689 on the Cullin-2 scaffold 11 . In its activated state, CRL2~N8 (the ~ stylization denotes a covalent interaction) recruits the ubiquitin-conjugated E2 enzyme via the RING domain of RBX1 12 . Ubiquitination now takes place, covalently adding ubiquitin to the substrate molecule docked at the CRL2 N-terminal. The activity of CRL2 is negatively regulated by the 331 kDa Constitutive Photomorphogenesis 9 Signalosome (CSN) complex, frequently referred to as the COP9 signalosome complex [13][14][15] . The CSN was originally identified as consisting of eight subunits

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Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

Cited by 83 publications

References 21 publications

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

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