Structure of Lipoprotein Lipase in Complex with GPIHBP1
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Structure of lipoprotein lipase in complex with GPIHBP1 Rishi Aroraa, Amitabh V. Nimonkarb, Daniel Bairda,1, Chunhua Wanga, Chun-Hao Chiua, Patricia A. Hortona, Susan Hanrahanb, Rose Cubbonb, Stephen Weldonc, William R. Tschantzc, Sascha Muellerd, Reto Brunnere, Philipp Lehrd, Peter Meierd, Johannes Ottle, Andrei Voznesenskyc, Pramod Pandeya,2, Thomas M. Smitha, Aleksandar Stojanovicd, Alec Flyerf, Timothy E. Bensona, Michael J. Romanowskia,3, and John W. Traugerb,3 aChemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139; bCardiovascular and Metabolic Disease Area, Novartis Institutes for Biomedical Research, Cambridge, MA 02139; cBiotherapeutic and Analytical Technologies, Novartis Institutes for Biomedical Research, Cambridge, MA 02139; dGlobal Discovery Chemistry, Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland; eChemical Biology and Therapeutics, 4002 Basel, Switzerland; and fGlobal Discovery Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA 02139 Edited by Ian A. Wilson, The Scripps Research Institute, La Jolla, CA, and approved April 16, 2019 (received for review November 27, 2018) Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) understanding of the structure and regulation of LPL will facil- metabolism. By catalyzing the hydrolysis of TGs present in TG-rich itate efforts to develop such therapeutics. lipoproteins (TRLs), LPL facilitates TG utilization and regulates Proper catabolism of TRL particles by LPL is dependent on the circulating TG and TRL concentrations. Until very recently, struc- accessory proteins LMF1 and GPIHBP1. Loss-of-function muta- tural information for LPL was limited to homology models, tions in LMF1 and GPIHBP1 strongly impair LPL activity and presumably due to the propensity of LPL to unfold and aggregate. cause severe hypertriglyceridemia (8–10). LMF1 is an endoplasmic By coexpressing LPL with a soluble variant of its accessory protein reticulum-resident chaperone protein that is required for proper glycosylphosphatidylinositol-anchored high-density lipoprotein folding and secretion of LPL (8). After LPL is secreted by paren- binding protein 1 (GPIHBP1) and with its chaperone protein lipase chymal cells of the heart, muscle, and adipose tissue, it initially binds maturation factor 1 (LMF1), we obtained a stable and homoge- to heparan sulfate proteoglycans (HSPG) in the subendothelial nous LPL/GPIHBP1 complex that was suitable for structure de- space. LPL subsequently binds to GPIHBP1, a membrane-bound termination. We report here X-ray crystal structures of human LPL protein expressed by capillary endothelial cells. GPIHBP1 trans- – in complex with human GPIHBP1 at 2.5 3.0 Å resolution, including ports LPL across the endothelial cell to the luminal side of a structure with a novel inhibitor bound to LPL. Binding of the the capillary endothelium where LPL binds to TRL particles in inhibitor resulted in ordering of the LPL lid and lipid-binding re- the bloodstream and catalyzes TRL-TG hydrolysis (10, 11). In the gions and thus enabled determination of the first crystal structure absence of functional GPIHBP1, LPL accumulates in the sub- of LPL that includes these important regions of the protein. It was endothelial space where it cannot interact with TRL particles (12). assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, Significance can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as LPL is the central enzyme in TG metabolism. We report here X-ray well as LPL stabilization and transport by GPIHBP1. crystal structures of LPL in complex with its accessory protein GPIHBP1, including a structure with a novel inhibitor bound in the X-ray crystallography | LPL | GPIHBP1 | lipase LPL active site. The inhibitor-bound structure includes the LPL lid and lipid-binding regions, which makes this the first complete Gs move through the bloodstream in TRL particles: very- crystal structure of LPL and provides insight into how these re- Tlow-density lipoproteins (VLDLs) transport TGs produced gions of LPL contribute to the recognition of TRL substrates. The by the liver, and chylomicrons transport TGs produced by the in- structural evidence and additional biochemical experiments pre- testines from dietary fat. LPL is expressed by TG-utilizing (heart sented here are consistent with a newly published report that and skeletal muscle) and TG-storing (adipose) tissues and catalyzes LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex and, therefore, help overturn the long-standing as- the hydrolysis of TGs present in TRL particles. The resulting free sumption that LPL is only active as a homodimer. fatty acids are taken up by the tissueandusedasanenergysource (in the heart and skeletal muscle) or for energy storage (in adipose), Author contributions: D.B., P.L., P.M., J.O., A.V., P.P., T.M.S., A.S., A.F., T.E.B., M.J.R., and and the resulting TRL remnants are cleared from circulation by the J.W.T. designed research; R.A., A.V.N., D.B., C.W., C.-H.C., P.A.H., S.H., R.C., S.W., W.R.T., liver or, in the case of some VLDL remnants, further metabolized S.M., R.B., P.L., and A.S. performed research; R.A., A.V.N., W.R.T., P.L., and J.O. analyzed into LDL by hepatic lipase (1). Hypertriglyceridemia (elevated data; and R.A., A.V.N., M.J.R., and J.W.T. wrote the paper. plasma TGs) is very common and is associated with an increased Conflict of interest statement: All authors are current or former employees of Novartis Institutes for Biomedical Research or Novartis Pharma AG, and some are also shareholders risk of myocardial infarction and other adverse cardiovascular events of Novartis. This work was funded by Novartis Institutes for Biomedical Research. (2). Human gene variants that increase LPL activity are associated This article is a PNAS Direct Submission. with lower plasma TG levels and reduced risk of coronary artery Published under the PNAS license. disease (3–7). A gain-of-function variant of LPL (S474X) that Data deposition: The atomic coordinates and structure factors have been deposited in the moderately increases secretion of the protein without affecting its Protein Data Bank, www.wwpdb.org (PDB ID codes 6OAZ, 6OAU, and 6OB0). catalytic activity is associated with lower circulating TG concentra- 1Present address: Casma Therapeutics, Cambridge, MA 02139. tions and lower risk of coronary artery disease in humans (3). Loss- 2Present address: Experimental and Chemical Biology, Merck Exploratory Science Center, of-function mutations in the LPL inhibitor proteins ANGPTL3 and Cambridge, MA 02141. ANGPTL4 are also associated with lower TGs and lower risk of 3To whom correspondence may be addressed. Email: [email protected] coronary artery disease in humans (3–7). These genetic association or [email protected]. studies highlight the potential for therapeutics that increase LPL This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activity to lower the risk of cardiovascular events in the large 1073/pnas.1820171116/-/DCSupplemental. population of people with hypertriglyceridemia. A thorough Published online May 9, 2019. 10360–10365 | PNAS | May 21, 2019 | vol. 116 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1820171116 Downloaded by guest on September 25, 2021 Structural homology modeling (13–15) and a recently reported multiangle light scattering (SEC-MALS) were consistent with the X-ray crystal structure of LPL bound to GPIHBP1 (16) indicate newly published report that LPL can be active as monomer (20). that LPL has a similar overall structure as pancreatic lipase (PL) Results and consists of a N-terminal catalytic domain (NTD) with an α/β-hydrolase fold and a C-terminal domain (CTD) with a Preparation of the LPL/sGPIHBP1 Complex. We initially expressed β-sandwich fold (13–16). In contrast to PL, which is monomeric, human LPL using transiently transfected HEK293-F cells with LPL has been reported to be homodimeric with a head-to-tail heparin added to the medium and purified LPL from the condi- orientation (15, 17–19). Furthermore, active LPL homodimers tioned medium by heparin affinity chromatography. LPL prepared have been reported to spontaneously dissociate into inactive in this manner was obtained with low yield (∼0.3 mg per liter of monomers (17, 19). In contrast, a new publication reevaluated culture), and SEC showed that the protein was aggregated. Al- these findings and concluded that LPL and LPL in complex with though this preparation of LPL was initially enzymatically active, it GPIHBP1 can be active as a monomer (20). GPIHBP1 has two completely lost activity after incubation at room temperature for domains—a N-terminal acidic domain in which most of the 10 h (SI Appendix,Fig.S1). Since GPIHBP1 had been reported to amino acids are aspartate or glutamate (21 of 26 amino acids in stabilize LPL (22, 23), we coexpressed His-tagged human LPL and + the human protein) and a three-fingered Ly6/uPAR (LU) do- human sGPIHBP1 and, after purification by Ni2 affinity chro- main. The LU domain is followed by a C-terminal GPI mem- matography followed by SEC, obtained a homogenous LPL/ brane anchor attachment sequence (10). Recombinant GPIHBP1 sGPIHBP1 complex with low yield (∼0.5 mg/L). Since LMF1 is with a C-terminal truncation that eliminates the GPI attachment necessary for proper LPL folding in vivo, we next coexpressed + site [soluble GPIHBP1 (sGPIHBP1)] has been used to show that humanLPL,sGPIHBP1,andLMF1andafterNi2 affinity chro- GPIHBP1 binds to LPL with high affinity, stabilizes LPL against matography followed by SEC obtained a homogenous LPL/ spontaneous unfolding, and interferes with the ability of ANGPTL3 sGPIHBP1 complex with good yield (∼6mg/L).Theenzymeac- and ANGPTL4 to inhibit LPL (21–23).