scholarly journals The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 49
Author(s):  
Kuhne ◽  
Bonnington ◽  
Malik ◽  
Thomann ◽  
Avenal ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Information ◽  
Binding Properties ◽  
Engineering Technology ◽  
Exchange Mass Spectrometry ◽  
Structural Impact ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
The Impact

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure–function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

scholarly journals Activation of GCN2 by the ribosomal P-stalk

2019 ◽  
Vol 116 (11) ◽  
pp. 4946-4954 ◽  
Author(s):  
Alison J. Inglis ◽  
Glenn R. Masson ◽  
Sichen Shao ◽  
Olga Perisic ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

scholarly journals The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

2021 ◽  
Vol 118 (12) ◽  
pp. e2026650118
Author(s):  
Katrine Z. Leth-Espensen ◽  
Kristian K. Kristensen ◽  
Anni Kumari ◽  
Anne-Marie L. Winther ◽  
Stephen G. Young ◽  
...  
Keyword(s):  
Lipoprotein Lipase ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Irreversible Unfolding ◽  
Deuterium Exchange Mass Spectrometry ◽  
Endothelial Receptor ◽  
Kinetics Of ◽  
Thermal Stability Of

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

scholarly journals Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

2019 ◽  
Author(s):  
Isaac Fisher ◽  
Meredith Jenkins ◽  
Greg Tall ◽  
John E Burke ◽  
Alan V. Smrcka
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
G Protein Coupled Receptors ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
G Protein Coupled ◽  
Inhibitory Interactions

AbstractPhospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

scholarly journals Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses

2021 ◽  
Author(s):  
Yimin Huang ◽  
Annalee W. Nguyen ◽  
Ching-Lin Hsieh ◽  
Rui Silva ◽  
Oladimeji S. Olaluwoye ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Conformational Epitope ◽  
Neutralizing Epitope ◽  
Highly Pathogenic ◽  
Exchange Mass Spectrometry ◽  
Global Pandemic ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Human Coronaviruses

ABSTRACTThree pathogenic human coronaviruses have emerged, with SARS-CoV-2 causing a global pandemic. While therapeutic antibodies targeting the SARS-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report three antibodies, binding epitopes conserved on the pre-fusion MERS, SARS-1 and SARS-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes in in vitro SARS-2 cell fusion and pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, consistent with binding to natural and engineered spike variants. This location at the spike trimer interface suggests 3A3 prevents the S2 conformational rearrangements required for virus-host cell fusion. This work defines a highly conserved vulnerable site on the SARS-2 S2 domain and may help guide the design of pan-protective spike immunogens.TEASERA conserved, neutralizing epitope in the S2 domain of coronavirus spike was identified as a target for pan-coronavirus therapy and vaccination.

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
Sign in / Sign up

Export Citation Format

Share Document