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

Abstract

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.

The Importance of Lipoprotein Lipase Regulation in Atherosclerosis

“…In contrast, the C-terminal PLAT domain is stable. The global unfolding observed with HDX-MS was confirmed by differential scanning fluorimetry (DSF) showing that the catalytic α/β-hydrolase domain unfolds with a T m of 34.8 • C, which is well below normal body temperature [17]. Binding of either GPIHBP1 or HSPGs stabilize the α/β-hydrolase domain profoundly (T m = 57.6 • C or 42.2 • C, respectively).…”

“…Studies with hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) provided the first details on the molecular mechanism(s) underlying this instability. The Achille's heel of the LPL protein weakening its thermal stability is found in the α/β-hydrolase domain, which unfolds spontaneously [17,20,53,62]. In contrast, the C-terminal PLAT domain is stable.…”

“…The molecular mechanism of ANGPTL4-mediated LPL inhibition represents a novel mode of action for enzyme inhibition. ANGPTL4 binds close to the active site in LPL and this binding event triggers the progressive and irreversible unfolding of LPL's α/β hydrolase domain [17,59,62,154]. Accordingly, ANGPTL4 was dubbed a "molecular unfolding chaperone" and its catalytic function is evident by the fact that sub-stoichiometric amounts of ANGPTL4 over time causes a complete LPL inhibition in vitro [59,154].…”

“…Accordingly, ANGPTL4 was dubbed a "molecular unfolding chaperone" and its catalytic function is evident by the fact that sub-stoichiometric amounts of ANGPTL4 over time causes a complete LPL inhibition in vitro [59,154]. Importantly, this irreversible inactivation by unfolding is mitigated by binding to GPIHBP1 [17,57,154,155].…”

“…Cartoon representation of the human LPL•GPIHBP1 complex and with the molecular surface of the LU domain (Ly6/uPAR protein domain family) of GPIHBP1shown as a transparent light blue envelope (generated with The PyMol Molecular Graphic System (Version 2.0 Schrödinger, LLC) using coordinates from the LPL•GPIHBP1 crystal structure; Protein Data Bank ID code 6E7K). LPL elements implicated in ANGPTL4 binding are highlighted in yellow[17]. GPIHBP1 binds to the C-terminal PLAT domain of LPL, and the location of GPIHBP1's membrane-tethering site (GPI anchor) is indicated[18].…”