Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

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Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

10.1101/2021.09.29.462278 ◽

2021 ◽

Author(s):

Jui-Hung Weng ◽

Phillop C. Aoto ◽

Robin Lorenz ◽

Jian Wu ◽

Sven H. Schmidt ◽

...

Keyword(s):

Md Simulations ◽

Kinase Domain ◽

Pharmacological Intervention ◽

Exchange Mass Spectrometry ◽

Allosteric Sites ◽

Hydrogen Deuterium ◽

Wd40 Domain ◽

Deuterium Exchange Mass Spectrometry ◽

And Function ◽

Hdx Ms

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson’s Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2 RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a “cap” that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

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Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

10.1101/681932 ◽

2019 ◽

Author(s):

Hanna S. Loving ◽

Eric S. Underbakke

Keyword(s):

Tyrosine Kinase ◽

Conformational Dynamics ◽

Kinase Domain ◽

Ferm Domain ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Protein Scaffolding ◽

Hdx Ms

AbstractPyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.Table of Contents graphic

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Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry

Bioinformatics ◽

10.1093/bioinformatics/btz022 ◽

2019 ◽

Vol 35 (17) ◽

pp. 3171-3173 ◽

Cited By ~ 26

Author(s):

Andy M C Lau ◽

Zainab Ahdash ◽

Chloe Martens ◽

Argyris Politis

Keyword(s):

Mass Spectrometry ◽

Conformational Dynamics ◽

Rapid Analysis ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Visualization Of Data ◽

Hdx Ms

Abstract Summary Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS. Availability and implementation Deuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license. Written in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library. According to the Wellcome Trust and UK research councils' Common Principles on Data Policy on data, software and materials management and sharing, all data supporting this study will be openly available from the software repository.

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Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry

10.1101/417998 ◽

2018 ◽

Cited By ~ 1

Author(s):

Andy M. C. Lau ◽

Zainab Ahdash ◽

Chloe Martens ◽

Argyris Politis

Keyword(s):

Mass Spectrometry ◽

Conformational Dynamics ◽

Rapid Analysis ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Visualization Of Data ◽

Hdx Ms

AbstractSummaryHydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS.AvailabilityDeuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license.Implementationwritten in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime [email protected]

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

eLife ◽

10.7554/elife.47402 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William John Allen ◽

Robin A Corey ◽

Ian Collinson ◽

...

Keyword(s):

Conformational Changes ◽

Opposite Effect ◽

Protein Transport ◽

Protein Translocation ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

The bacterial Sec translocon is a multi-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 pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.

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PyHDX: Derivation and visualization of protection factors from Hydrogen-Deuterium Exchange Mass Spectrometry at near residue resolution

10.1101/2020.09.30.320887 ◽

2020 ◽

Author(s):

Jochem H. Smit ◽

Srinath Krishnamurthy ◽

Bindu Y. Srinivasu ◽

Spyridoula Karamanou ◽

Anastassios Economou

Keyword(s):

Mass Spectrometry ◽

Local Equilibrium ◽

Conformational Dynamics ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Free Energies ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

AbstractHydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful technique to monitor the intrinsic and conformational dynamics of proteins. Most HDX-MS experiments compare protein states (e.g. apoprotein vs liganded) and provide detailed information on differential dynamics between them obtained from multiple overlapping peptides. However, differential dynamics are difficult to compare across protein derivatives, oligomeric assemblies, homologues and samples treated under different buffer and protease conditions. A main reason is that peptide-based D-uptake differences do not inform on absolute intrinsic dynamics at the level of single aminoacyl residues. Such information is offered by protection factors, i.e. the position of the local equilibrium between the D-exchange-competent ‘open’ state and the non-exchanging ‘closed’ state. We present PyHDX, a software tool to calculate protection factors and Gibbs free energies typically within minutes from HDX-MS-derived peptide lists. PyHDX provides intrinsic information on the thermodynamics of protein dynamics at single-residue level. An interactive web interface further streamlines the process of transforming peptide lists to either coloured linear sequence maps or 3D structures of Gibbs free energies/protection factors.AvailabilityPyHDX source code is released under the MIT license and can be accessed at the project’s GitHub page.

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HDX-MS: An Analytical Tool to Capture Protein Motion in Action

Biomedicines ◽

10.3390/biomedicines8070224 ◽

2020 ◽

Vol 8 (7) ◽

pp. 224

Author(s):

Dominic Narang ◽

Cristina Lento ◽

Derek J. Wilson

Keyword(s):

Protein Dynamics ◽

Vaccine Development ◽

Conformational Dynamics ◽

X Ray Crystallography ◽

Exchange Mass Spectrometry ◽

Protein Functions ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Biophysical Techniques ◽

Hdx Ms

Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

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