scholarly journals The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Simon Mysling ◽  
Kristian Kølby Kristensen ◽  
Mikael Larsson ◽  
Anne P Beigneux ◽  
Henrik Gårdsvoll ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipoprotein Lipase ◽  
Catalytic Domain ◽  
Exchange Mass Spectrometry ◽  
Intrinsically Disordered ◽  
Acidic Domain ◽  
Terminal Domain ◽  
Capillary Endothelial Cells ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

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 A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase

2018 ◽  
Vol 115 (26) ◽  
pp. E6020-E6029 ◽  
Author(s):  
Kristian K. Kristensen ◽  
Søren Roi Midtgaard ◽  
Simon Mysling ◽  
Oleg Kovrov ◽  
Lars Bo Hansen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lipoprotein Lipase ◽  
Catalytic Domain ◽  
Intrinsically Disordered ◽  
Severe Hypertriglyceridemia ◽  
Biophysical Studies ◽  
Close Proximity ◽  
Key Factor ◽  
Capillary Endothelial Cells ◽  
Structure And Activity

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL’s catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1’s IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1–LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1–LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1’s IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1’s ability to preserve LPL structure and activity.

scholarly journals ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

2019 ◽  
Author(s):  
Xiaoshan Shi ◽  
Adam L. Yokom ◽  
Chunxin Wang ◽  
Lindsey N. Young ◽  
Richard J. Youle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Exchange Mass Spectrometry ◽  
Terminal Domain ◽  
Negative Stain ◽  
Complex Organization ◽  
Hydrogen Deuterium ◽  
Negative Stain Electron Microscopy ◽  
Deuterium Exchange Mass Spectrometry ◽  
Structure Similarity ◽  
20 Nm

AbstractThe autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain electron microscopy (EM) and multiangle light scattering showed that FIP200 is a dimer whilst a single molecule each of the other subunits is present. The FIP200 NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13-ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structure similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy initiating TBK1 kinase and the ULK1 kinase complex.SummaryThe human ULK complex consists of ULK1/2, FIP200, ATG13, and ATG101. We found that the FIP200 N-terminal domain is a C-shaped dimer that binds directly to a single ATG13 molecule and serves as the organizing hub of the complex.

scholarly journals Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Salvador F. Ausar ◽  
Shaolong Zhu ◽  
Jessica Duprez ◽  
Michael Cohen ◽  
Thomas Bertrand ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Catalytic Domain ◽  
Mass Cytometry ◽  
Subunit Interactions ◽  
Dynamic Changes ◽  
Robust Immune Response ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
The Impact

AbstractThe mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD+ binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.

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