Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

Abstract

Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and highresolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange massspectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding. FIGURE 1 | Schematic overview of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) workflow. Proteins are labeled in D 2 O buffer for a predefined time, followed by exchange quenching and proteolysis. The peptides are separated, and their mass is detected using MS. Finally, the amount of incorporated deuterium within each peptide is calculated for each time point. Giladi and Khananshvili HDX-MS of Membrane Transporters Frontiers in Pharmacology | www.frontiersin.org

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…There is a plethora of examples for the application of HDX-MS in protein interaction studies [ 21 , 106 ]. More challenging are those applications, where the protein is part of a large protein complex, such as a nuclear pore complex [ 34 , 35 ], embedded in a membrane [ 15 , 107 ], or disordered [ 108 , 109 ]. Selected protein modifications, like glycosylation and disulfide bonding, hinder proteolysis and/or mass analysis [ 26 ].…”

“…Last year, a group of eminent researchers in the field formulated clear recommendations for performing and interpreting HDX-MS experiments [ 4 ] and hence paved the way towards truly standardized experiments. The applications of HDX-MS are very versatile: they include studies of protein complexes [ 5 , 6 , 7 , 8 , 9 , 10 ], ligand binding [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], dynamic properties like conformational changes and folding/unfolding/refolding [ 26 ], conformational changes that arise from allosteric effects [ 27 , 28 , 29 ], structure and stability of biopharmaceuticals [ 17 , 30 , 31 ] and epitopes [ 15 , 32 , 33 ], etc. Unlike several other biophysical techniques, HDX-MS possesses the advantages of no or very high size limit [ 8 ] and is useful for studying individual proteins as well as large complexes [ 34 , 35 ].…”

Current Methods for Detecting Cell Membrane Transient Interactions

“…Furthermore, it is challenging to apply NMR and MS to study really fast (< ms) membrane interactions or conformational changes. Indeed, efforts have been taken to improve the temporal resolution of MS, for example, by conjugating with a flow quenching system (Rist et al, 2005 ; Giladi and Khananshvili, 2020 ).…”

“…MS does not provide high-resolution target structure, but it can measure the mass and stoichiometry of the complex (Pyle et al, 2018 ; Frick and Schmidt, 2019 ). Among different types of MS, hydrogen–deuterium exchange MS (HDX-MS) has gained much attention for studying transient (millisecond range) protein conformational changes (Rist et al, 2005 ; Giladi and Khananshvili, 2020 ). For example, HDX-MS has been used to study how transient lipid–protein interactions can induce conformational and functional changes of bacterial leucine transporter LeuT (Adhikary et al, 2017 ), rhomboid protease GlpG (Reading et al, 2017 ), and various secondary transporters (Martens et al, 2018 ).…”

“…In HDX-MS, labile hydrogens in the protein complex can be exchanged with deuterium. The mass uptake at different locations of the complex will then be measured and mapped to allow visualization of its structural dynamics ( Figure 2B ; Martens et al, 2019 ; Giladi and Khananshvili, 2020 ). High-purity samples are desirable but not essential, because the contaminants can be easily ruled out during data processing (Martens et al, 2019 ).…”