scholarly journals Expression and purification of Protease Activated Receptor 4 (PAR4) and analysis with histidine hydrogen deuterium exchange

2019 ◽  
Author(s):  
Maria de la Fuente ◽  
Xu Han ◽  
Masaru Miyagi ◽  
Marvin T. Nieman
Keyword(s):  
Family Member ◽  
Conformational Changes ◽  
Deuterium Exchange ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Hydrogen Deuterium Exchange ◽  
Expression And Purification ◽  
Agonist Peptide ◽  
Hydrogen Deuterium ◽  
Tethered Ligand

ABSTRACTProtease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolyis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine-hydrogen deuterium exchange (His-HDX). PAR4 has 9 histidines that are spaced throughout the protein allowing a global view of solvent accessible and non-accessible regions. Peptides containing each of the 9 His residues were used to determine the t1/2 for each His residue in apo or thrombin activated PAR4. The thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin cleaved PAR4 also was resistant to thermolysin digestion. In contrast, activation with the PAR4 agonist peptide was digested at the same rate as apo PAR4. Further analysis showed the C-terminus is protected in thrombin activated PAR4 compared to uncleaved or agonist peptide treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

Analysis of Protease Activated Receptor 4 (PAR4) By Histidine Hydrogen Deuterium Exchange

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4166-4166
Author(s):  
Marvin T. Nieman ◽  
Maria de la Fuente ◽  
Masaru Miyagi
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Hydrogen Deuterium Exchange ◽  
Ligand Activation ◽  
Histidine Residues ◽  
Biophysical Studies ◽  
Hydrogen Deuterium ◽  
Tethered Ligand

Abstract Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered ligand that interacts with a binding pocket on the receptor. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. We now describe the first biophysical studies examining this unique mode of activation using PAR4. PAR4 is one of the thrombin receptors on platelets and is required for sustained platelet activation. Recent data from our laboratory and others suggest that PAR4 has the potential to be a therapeutic target. To examine the tethered ligand activation mechanism in general and further examine PAR4 as a target, we have expressed and purified PAR4 that does not contain additional stabilizing sequences from Sf9 cells for biophysical studies. The current study uses histidine-hydrogen deuterium exchange (His-HDX). His-HDX is a structural mass spectrometry technique that relies on the slow exchange of the C2 carbon on the imidazole ring of histidine. PAR4 has nine histidine residues that are well spaced throughout the protein, which gives us a global view of solvent accessible and non-accessible regions. Unique peptides were generated that contained each of the histidine residues (His136, His159, His180, His229, His240, His269, His280, His306, and His380). These peptides were used to determine the t1/2 for each His residue in full length or thrombin cleaved PAR4. The t1/2 values for His159 and His269 were greater than 40 hours in both states indicating that these two histidine residues are not exposed to solvent in either case. In contrast, the thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have a decrease in solvent accessibility upon thrombin treatment. These residues are spread throughout the protein, which indicates that PAR4 undergoes a global conformational change and the overall structure becomes more rigid. In agreement with these data, thrombin cleaved PAR4 is more resistant to protease digestion by chymotrypsin. The half-life was extended 4-fold over the uncleaved PAR4. We are also able to make conclusions regarding specific regions of PAR4. His229 is located in extracellular loop 2 near the proposed ligand-binding site and has a 2-fold increase in t1/2 following thrombin treatment. These data suggest that the tethered ligand may be binding to this region and protecting it from exchange. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysical approaches. These studies shed light on the overall conformational changes that follow activation of PARs by a protease. Finally, the His-HDX used for purified PAR4 lays important groundwork that will allow us to examine PAR4 and other platelet GPCRs in their native membrane environment using this and related mass spectrometry approaches. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hydrogen Exchange Mass Spectrometry Reveals Allosteric Interactions Between the Distal and the Proximal Domains of Human ADAMTS13

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2535-2535 ◽  
Author(s):  
Catherine B. Zander ◽  
Leland Mayne ◽  
S. Walter Englander ◽  
X. Long Zheng
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Hydrogen Exchange ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Allosteric Interaction ◽  
Allosteric Interactions ◽  
Hydrogen Deuterium ◽  
Direct Binding ◽  
Terminal Domains

Abstract Background: Deletion of the distal C-terminal domains, or a decrease in pH, or the binding of a monoclonal antibody to the C-terminal domains of ADAMTS13 increases its proteolytic activity towards VWF peptide substrate, VWF73, by approximately 2-fold; this suggests an allosteric interaction between the distal and proximal domains of ADAMTS13. However, neither the underlying mechanism of the allosteric interaction nor the exact sites of interaction between the proximal domains and the distal C-terminal domain are fully understood. Objective: To determine the binding sites and kinetic interactions between the proximal and distal domains of ADAMTS13 and to gain insight into the mechanism of allosteric regulation of ADAMTS13 function. Methods: Hydrogen deuterium exchange plus LC-mass spectrometry (HX-MS) and surface plasmon resonance (SPR) were employed to determine conformational changes and direct binding interactions between the proximal and distal domains of ADAMTS13. Results: Recombinant full-length (rFL-A13) and an ADAMTS13 variant truncated after the spacer domain (rMDTCS) were purified to homogeneity. After incubation with deuterium for various times (0-6 hours), both rFL-A13 and rMDTCS were digested with pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Overall, 188 of the total 338 peptides in rFL-A13 were identified, spanning the metalloprotease, cysteine-rich, and spacer domain. Only 16 peptides were found to be less accessible (or protected from deuterium-hydrogen exchange) in rFL-A13 than in rMDTCS. Two of these regions reside in the metalloprotease domain (residue 78-127 and 282-304). The next region is at a loop on the cysteine-rich domain (residue 446-473 and 495-501). The next peptides are in the adjacent loops of the spacer domain with one peptide 596-603, and five overlapping peptides spanning from 632-642, and then finally one peptide from the C-terminal of the MDTCS variant (residue 676 to 688). These results indicate that the distal C-terminal domains interact with multiple discrete sites in the proximal domains of ADAMTS13, resulting in protection from hydrogen-deuterium exchange. Such an interaction was further demonstrated by direct binding experiments with SPR. The distal domains including CUB, T2-CUB, and T5-CUB bound directly to M, MDT, and MDTCS under flow with high affinity. Conclusion: Our findings demonstrate the allosteric interactions between the distal domains and the proximal domains at multiple discrete sites. Such an interaction may regulate catalytic efficiency and substrate specificity of the metalloprotease domain of ADAMTS13 under various conditions. Disclosures Zheng: Ablynx: Consultancy; Alexion: Research Funding.

scholarly journals Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

Biochemistry ◽  
2017 ◽  
Vol 56 (21) ◽  
pp. 2676-2689 ◽  
Author(s):  
Sharlyn J. Mazur ◽  
Elyssia S. Gallagher ◽  
Subrata Debnath ◽  
Stewart R. Durell ◽  
Kyle W. Anderson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

scholarly journals Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange

2004 ◽  
Vol 382 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Frantz SCHEIRLINCKX ◽  
Vincent RAUSSENS ◽  
Jean-Marie RUYSSCHAERT ◽  
Erik GOORMAGHTIGH
Keyword(s):  
Fourier Transform ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Structural Information ◽  
Deuterium Exchange ◽  
Attenuated Total Reflection ◽  
Hydrogen Deuterium Exchange ◽  
Hydrogen Deuterium ◽  
Difference Fourier

Gastric H+/K+-ATPase is a P-type ATPase responsible for acid secretion in the stomach. This protein adopts mainly two conformations called E1 and E2. Even though two high-resolution structures for a P-ATPase in these conformations are available, little structural information is available about the transition between these two conformations. In the present study, we used two experimental approaches to investigate the structural differences that occur when gastric ATPase is placed in the presence of various ligands and ligand combinations. We used attenuated total reflection–Fourier-transform IR experiments under a flowing buffer to modify the environment of the protein inside the measurement cell. The high accuracy of the results allowed us to demonstrate that the E1–E2 transition induces a net change in the secondary structure that concerns 10–15 amino acid residues of a total of 1324 in the proteins. The E2.K+ structure is characterized by a decreased β-sheet content and an increase in the disordered structure content with respect to the E1 form of the enzyme. Modifications in the absorption of the side chain of amino acids are also suggested. By using hydrogen/deuterium-exchange kinetics, we show that tertiary-structure modifications occurred in the presence of the same ligands, but these changes involved several hundreds of residues. The present study suggests that conformational changes in the catalytic cycle imply secondary-structure rearrangements of small hinge regions that have an impact on large domain re-organizations.

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