Ligand-induced conformational dynamics of the
 Escherichia coli
 Na
 +
 /H
 +
 antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry

Eisinger, Martin Lorenz; Dörrbaum, Aline Ricarda; Michel, Hartmut; Padan, Etana; Langer, Julian D.

doi:10.1073/pnas.1703422114

Cited by 42 publications

(46 citation statements)

References 60 publications

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“…8, D and E). Surface residues on both PDZ domains displayed higher deuteration on a short time scale, consistent with the view that solvent accessibility in the crystal structure correlates with the relative deuterium uptake (63). Strikingly, the ␤-sheets in PDZ2 (Fig.…”

Section: Structural Determinants Of Nherf1 Binding To Npt2a and Confosupporting

confidence: 83%

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

Zhang

Xiao

Paredes

et al. 2019

Journal of Biological Chemistry

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Na ؉ -H ؉ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodiumphosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1␣ (PP1␣), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 . Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1␣-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ␤-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally . 4 The abbreviations used are: PTH, parathyroid hormone; PTHR, PTH receptor; NHERF1, Na ϩ /H ϩ -exchanger regulatory factor 1; GnTI Ϫ , N-acetylglucosaminyltransferase-deficient HEK-293S cells; TAP-NHERF1, tandem affinity purification-tagged NHERF1; FLIM, fluorescence lifetime imaging; HDX, hydrogen/deuterium exchange mass spectrometry; pSer 290 , phosphorylated Ser 290 ; NP40, Nonidet P-40; TAMRA, carboxytetramethylrhodamine; SILAC, stable isotope labeling of amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ND, nondeuterated; FD, fully deuterated; EBD, ezrin-binding domain; PDB, Protein Data Bank; 2Me-4OMe-TM, 7-hydroxy-5,5-dimethyl-10-(4-methoxy-2-methylphenyl)-dibenzo-[b,e]-silin-3(5H)-one; ANOVA, analysis of variance; ID, intrinsically disordered; TTN, tautomycetin; RPTEC, renal proximal tubule epithelial cell; pen/strep, penicillin and streptomycin; CFP, cyan fluorescent protein; FERM, Ezrin-Radixin-Moesin; MD, molecular dynamics.

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Section: Structural Determinants Of Nherf1 Binding To Npt2a and Confosupporting

confidence: 83%

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

Zhang

Xiao

Paredes

et al. 2019

Journal of Biological Chemistry

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“…The main structural model used to study Nha orthologs is Escherichia coli NhaA, for which a crystallographic structure of the inactive state at acidic pH is available (Hunte et al, 2005). To study the structural dynamics of NhaA under physiological pH, HDX-MS was recently used (Eisinger et al, 2017). Crucial to the insights obtained in this study, HDX-MS provides global dynamic data, in contrast to methods based on site-specific labeling, which only detect movements of pre-defined protein regions.…”

Section: Conformational Transitions Underlyingmentioning

confidence: 99%

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

Giladi

Khananshvili

2020

Front. Pharmacol.

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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

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“…For example, the hydrophobic transmembrane segments of MPs can be inherently difficult to digest and observe by LC-MS, although this is often protein and digestion condition dependent [189], and does not always preclude structural analyses. Recent studies on detergent and lipid solubilized proteins have demonstrated that both are compatible with HDX-MS [181,[190][191][192][193][194][195].…”

Section: Hydrogen/deuterium Exchangementioning

confidence: 99%

“…8) [190]. The conformational changes in green cone opsin upon light activation have also been studied [196], as well as the alternating access mechanism of the Na + /H + antiporter NhaA [191].…”

Section: Hydrogen/deuterium Exchangementioning

confidence: 99%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

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Ligand-induced conformational dynamics of the Escherichia coli Na ⁺ /H ⁺ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry

Cited by 42 publications

References 60 publications

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

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

Mass spectrometry-enabled structural biology of membrane proteins

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Ligand-induced conformational dynamics of the Escherichia coli Na + /H + antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry

Cited by 42 publications

References 60 publications

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

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

Mass spectrometry-enabled structural biology of membrane proteins

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Product

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About

Ligand-induced conformational dynamics of the Escherichia coli Na ⁺ /H ⁺ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry