Different conformational dynamics of PDZ1 and PDZ2 in full-length EBP50 analyzed by hydrogen/deuterium exchange mass spectrometry

2015 ◽  
Vol 93 (4) ◽  
pp. 290-297 ◽  
Author(s):  
Ji Young Park ◽  
Nguyen Minh Duc ◽  
Dong Kyun Kim ◽  
Su Youn Lee ◽  
Sheng Li ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Full Length ◽  
Hydrogen Deuterium Exchange ◽  
Pdz Domains ◽  
Ligand Selectivity ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein expressed in polarized epithelial cells in various organs, including the liver, kidney, and small intestine, in which it regulates the trafficking and targeting cellular proteins. EBP50 contains two postsynaptic density-95/disk-large/ZO-1 homology (PDZ) domains (e.g., PDZ1 and PDZ2) and an ezrin/radixin/moesin-binding (EB) domain. PDZ domains are one of the major scaffolding domains regulating protein–protein interactions with critical biological roles in cell polarity, migration, proliferation, recognition, and cell–cell interaction. PDZ1 and PDZ2 in EBP50 have different ligand selectivity, although several high-resolution structural studies of isolated PDZ1 and PDZ2 showed similar structures. We studied the conformations of full-length EBP50 and isolated PDZ1 and PDZ2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The deuterium uptake profiles of isolated PDZ1 and PDZ2 were similar to those of full-length EBP50. Interestingly, PDZ1 was more dynamic than PDZ2, and these PDZ domains underwent different conformational changes upon ligand binding. These results might explain the differences in ligand-selectivity between PDZ1 and PDZ2.

Conformational Dynamics Analysis of MEK1 Using Hydrogen/Deuterium Exchange Mass Spectrometry

2020 ◽  
Vol 27 ◽  
Author(s):  
Min Woo Yun ◽  
Kiae Kim ◽  
Ji Young Park ◽  
Ka Young Chung
Keyword(s):  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Full Length ◽  
Atp Binding ◽  
Hydrogen Deuterium Exchange ◽  
Mapk Kinase ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Background: Activation of mitogen-activated protein kinases (MAPKs) is regulated by a phosphorylation cascade comprising three kinases, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP2K1 and MAPK2K2, also known as MEK1 and MEK2, activate ERK1 and ERK2. The structure of the MAPK signaling cascade has been studied, but high-resolution structural studies of MAP2Ks have often focused on kinase domains or docking sites, but not on full-length proteins. Objective: To understand the conformational dynamics of MEK1. Methods: Full-length MEK1 was purified from Escherichia coli (BL21), and its conformational dynamics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The effects of ATP binding were examined by coincubating MEK1 and adenylyl-imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analog. Results: MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through β1, αI, and the C-terminal helix. AMP-PNP binding was found to reduce conformational dynamics within the glycine-rich loop and regions near the DFG motif, along with the activation lip. Conclusion: We report for the first time that MEK1 has regions that slowly change its folded and unfolded states (mixed EX1/EX2 kinetics) and also report the conformational effects of ATP-binding to MEK1.

scholarly journals Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

2016 ◽  
Vol 113 (9) ◽  
pp. 2412-2417 ◽  
Author(s):  
Siavash Vahidi ◽  
Yumin Bi ◽  
Stanley D. Dunn ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Molecular Motor ◽  
Conformational Dynamics ◽  
Bound State ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Membrane Bound ◽  
Deuterium Exchange Mass Spectrometry

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

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

2017 ◽  
Vol 114 (44) ◽  
pp. 11691-11696 ◽  
Author(s):  
Martin Lorenz Eisinger ◽  
Aline Ricarda Dörrbaum ◽  
Hartmut Michel ◽  
Etana Padan ◽  
Julian David Langer
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Conformational Dynamics ◽  
Ion Homeostasis ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Ion Translocation ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

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