Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan 29;116(5):1723-1732.
doi: 10.1073/pnas.1817984116. Epub 2018 Dec 17.

Structure of the lipoprotein lipase-GPIHBP1 complex that mediates plasma triglyceride hydrolysis

Gabriel Birrane  1 , Anne P Beigneux  2 , Brian Dwyer  3 , Bettina Strack-Logue  3 , Kristian Kølby Kristensen  4   5 , Omar L Francone  3 , Loren G Fong  2 , Haydyn D T Mertens  6 , Clark Q Pan  3 , Michael Ploug  4   5 , Stephen G Young  7 , Muthuraman Meiyappan  8
Affiliations

Structure of the lipoprotein lipase-GPIHBP1 complex that mediates plasma triglyceride hydrolysis

Gabriel Birrane et al. Proc Natl Acad Sci U S A. .

Abstract

Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.

Keywords: GPIHBP1; X-ray crystallography; lipase; lipoproteins; triglycerides.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: B.D., B.S.-L., C.Q.P., and M.M. are employees of Shire and hold stock and stock options in Shire. R.Z. and S.G.Y. are coordinators on a Leducq Transatlantic Network Grant. They have not collaborated directly on this project.

Figures

Fig. 1.
Fig. 1.
Human GPIHBP1 binds to LPL with 1:1 stoichiometry, as judged by native polyacrylamide gel electrophoresis. In the absence of GPIHBP1, little LPL enters native polyacrylamide gels and does not migrate as a distinct band, simply because LPL is a very basic protein. GPIHBP1 is highly acidic; thus, an LPL–GPIHBP1 complex readily enters native gels and migrates as a distinct band. (A) Binding stoichiometry of LPL and GPIHBP1, determined by titrating 1.5 µM human LPL (lane 1) with increasing amounts of full-length human GPIHBP1 [0.3–3.0 µM full-length GPIHBP1 (GPIHBP121–151)] (lanes 2–11). The relative amounts of LPL–GPIHBP1 complexes (determined by scanning Coomassie blue-stained bands) are superimposed on the gels as black diamonds. These studies reveal a 1:1 binding stoichiometry. (B) A similar titration of 7.6 µM LPL (lane 1) with increasing amounts of a synthetic peptide corresponding to the N-terminal acidic domain of GPIHBP1 (GPIHBP121–53; 1–10 µM) (lanes 2–11). These experiments reveal 1:1 binding stoichiometry.
Fig. 2.
Fig. 2.
Structure of the LPL–GPIHBP1 complex, as depicted by ribbon representations of the two LPL–GPIHBP1 complexes in the asymmetric crystallographic unit. LPL (purple) has five disulfide bonds (C54–C67, C243–C266, C291–C302, C305–C310, and C445–C465); a single calcium ion (orange sphere) coordinated by A194, R197, S199, D201, and D202; and two N-linked glycans (at N70 and N386). GPIHBP1 (green) has one N-linked glycan (at N78). LPL contains an N-terminal α/β-hydrolase domain (N) containing 6 α-helices and 10 β-strands and a C-terminal flattened β-barrel domain (C) containing 12 β-strands, connected by a hinge region. The numbering of the β-strands in GPIHBP1 follows the nomenclature proposed for LU domain proteins (55). IDR, intrinsically disordered region.
Fig. 3.
Fig. 3.
Small-angle X-ray scattering (SAXS) analysis of the LPL–GPIHBP1 complex in solution. (A) SAXS data (gray circles) and the fit of the ab initio model reconstructed using DAMMIF with P2 symmetry (green line). The Lower panel shows the error-weighted residual differences between the model fit and the experimental data. (B) The real-space distance–distribution of the SAXS data and model. The Inset shows the superposition of the crystal structure of the LPL–GPIHBP1 complex (PDB ID code 6E7K) with the filtered average ab initio model.
Fig. 4.
Fig. 4.
LPL–GPIHBP1 interactions. GPIHBP1 (green sticks, showing Cα positions and key side chains) forms a concave hydrophobic surface. That surface interacts with the C-terminal domain of LPL (purple sticks, showing key LPL side chains). A semitransparent surface of LPL is colored according to electrostatic potential [red (acidic, −5 kBT), white (neutral, 0 kBT), and blue (basic, 5 kBT)]. Electrostatic potentials were calculated with DelPhi (56). All three fingers of GPIHBP1, but particularly fingers 2 and 3, interact with LPL, largely by hydrophobic contacts.
Fig. 5.
Fig. 5.
Distinct position for GPIHBP1 binding to LPL compared with colipase binding to pancreatic lipase. The structure of the LPL–GPIHBP1 complex (C-terminal domain of LPL in purple; GPIHBP1 in green) is superimposed on that of the pancreatic lipase (PL)–colipase complex (C-terminal domain of PL in khaki; colipase in cyan), revealing that the binding sites for GPIHBP1 and colipase on the C-terminal domains of their partner lipase (LPL and PL, respectively) are distinct. Colipase binds by polar interactions to one face of the flattened β-barrel via two hairpin loops (22), whereas GPIHBP1 interacts with both faces through a large hydrophobic interface. Both GPIHBP1 and colipase are small multifingered proteins with five disulfide bonds; however, the two proteins are evolutionarily distinct, and the arrangement of the disulfide bonds in the two proteins is different.
Fig. 6.
Fig. 6.
Electrostatic surface of LPL showing the site for GPIHBP1 binding. GPIHBP1’s LU domain forms a concave hydrophobic surface that interacts with a hydrophobic surface on the C-terminal domain (CTD) of LPL (image on Right). LPL has a single large, contiguous basic patch (∼2,400 Å2) spanning the CTD, the hinge region, and the N-terminal catalytic domain (Middle image). The C terminus of GPIHBP1, where the GPI anchor would be attached, and the N terminus of GPIHBP1, from which GPIHBP1’s acidic intrinsically disordered region (IDR) projects, are indicated by arrows (Middle and Right images). GPIHBP1’s acidic IDR, >60 Å in length (10), is expected to project across and interact transiently with LPL’s large basic patch (Middle image). The sequence of GPIHBP1’s highly acidic IDR is shown in SI Appendix, Fig. S2. In the image on the Left, the catalytic pocket is highlighted by a yellow dotted circle. The Trp motif and lid sequences are depicted as dashed green and cyan lines, respectively. Electrostatic potentials were calculated as in Fig. 4.
Fig. 7.
Fig. 7.
Schematic diagrams depicting interactions between M404 in LPL and GPIHBP1 and between W109 in GPIHBP1 and LPL. (A) Interactions of M404 in LPL (purple) with a hydrophobic pocket formed by GPIHBP1 (green) (GPIHBP1 residues V121, E122, T124, and V126; all in finger 3 of GPIHBP1’s LU domain). Replacing M404 with an arginine disrupts binding of LPL to GPIHBP1 (Fig. 8). (B) Hydrophobic interactions between W109 in GPIHBP1 and LPL. GPIHBP1-W109 is located in a hydrophobic pocket and interacts with multiple LPL residues (Dataset S1). This figure depicts the C445–C465 disulfide bond in LPL but does not show all of the hydrophobic interactions between those two cysteines and GPIHBP1. LPL-C445 interacts with GPIHBP1 residues K69 and S70; LPL-C465 interacts with multiple GPIHBP1 residues (Dataset S1). A p.C445Y mutation in LPL abolishes binding of LPL to GPIHBP1 (17), likely by disrupting the conformation of the loop established by the C445–465 disulfide bond.
Fig. 8.
Fig. 8.
Testing the impact of an LPL missense mutation (p.M404R) on GPIHBP1 binding. (A) CHO cells that had been transfected with S-protein–tagged versions of wild-type (wt) human GPIHBP1 or GPIHBP1-W109S (or empty vector) were coplated with cells that had been transfected with V5-tagged versions of human LPL-wt, LPL-M404R, or LPL-C445Y. The p.W109S mutation in GPIHBP1 and the p.C445Y mutation in LPL interfere with LPL–GPIHBP1 interactions (17, 35). The ability of LPL to bind to GPIHBP1 on the surface of cells was assessed by immunocytochemistry. Freshly secreted LPL-wt (green) was captured by GPIHBP1 (red), resulting in colocalization of LPL and GPIHBP1 in the merged image. GPIHBP1-W109S has no capacity to bind LPL-wt; consequently, no colocalization was observed on the merged image. LPL-M404R had little or no capacity to bind to GPIHBP1 (no colocalization on the merged image). (B) LPL activity and mass assays revealed that LPL-M404R is catalytically active but binds poorly to GPIHBP1. Fresh medium from CHO cells that had been transfected with FLAG-tagged human LPL-wt, LPL-M404R, or LPL-C445Y was added to wells of a 96-well ELISA plate that had been coated with a FLAG-specific antibody (FLAG-Ab) or with human GPIHBP1. Relative amounts of LPL mass were assessed with an HRP-labeled monoclonal antibody against LPL (5D2) and plotted as optical density (OD). LPL-M404R was efficiently captured by the FLAG antibody, but little LPL-M404R was captured by GPIHBP1. LPL-M404R binding to GPIHBP1 was reduced by 79% and 85% in two independent experiments (compared with LPL-wt binding to GPIHBP1). Triglyceride hydrolase activity of LPL captured on FLAG antibody-coated wells was assessed with [3H]triolein as the substrate. LPL-wt, LPL-M404R, and LPL-C445Y were catalytically active. (C) Assessing LPL-M404R catalytic activity and secretion from cells. CHO cells were transfected with V5-tagged versions of LPL-wt, LPL-D201V, LPL-M404R, or LPL-S159G (mutation of the critical serine in LPL’s catalytic triad). The LPL in cell lysates and media was assessed by Western blotting with a V5 antibody (green). Actin (red) was used as a loading control. LPL activity in the medium was assessed with a [3H]triolein substrate. LPL-M404R was synthesized and secreted by CHO cells, and there was robust LPL-M404R catalytic activity in the cell culture medium. The enzymatic activity of LPL-M404R was 103%, 110%, 115%, and 103% of the activity of LPL-wt in four independent experiments.
Fig. 9.
Fig. 9.
The calcium binding site in human LPL. (A) Stick representation of LPL’s Ca2+ binding site, showing main-chain and side-chain hydrogen bonding (dashed yellow lines). Difference electron density maps were contoured at 7σ (blue) and 2.8σ (cyan) for the calcium atom and water (w) molecules, respectively. (B) Assessing the impact of mutations in calcium-coordinating amino acids on LPL secretion from cells. CHO cells were transfected with FLAG-tagged versions of wild-type (wt) human LPL and several mutant LPLs (D201V, D201E, D202E, and S159G). LPL-S159G is an inactivating mutation in LPL’s catalytic triad. A Western blot with a FLAG-specific antibody (green) was used to detect LPL in cell lysates and the cell culture medium. Actin (red) was used as a loading control. (C) LPL activity in the medium of LPL-transfected cells. LPL activity was assessed with a [3H]triolein substrate (triglyceride hydrolase activity) and a DGGR substrate (esterase activity). The catalytic activities of the different LPL proteins are plotted as a percentage of those observed with LPL-wt (set at 100%). Based on the structure of LPL, we predicted that replacing D201 with a glutamate would not interfere with calcium coordination, whereas replacing D202 with a glutamate would disrupt calcium binding. D202 is buried and the extra methylene in a Glu mutation would not allow calcium coordination. D201 is located on the surface, and a Glu in that position retains the ability to coordinate calcium. LPL-D201V activity was 10.5%, 6.1%, 4.1%, and 12.9% of LPL-wt in four independent experiments; LPL-D201E activity was 100% and 127% of LPL-wt in two independent experiments; LPL-D202E activity was 1.6% and 0.0% of control in two independent experiments.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Korn ED. Clearing factor, a heparin-activated lipoprotein lipase. II. Substrate specificity and activation of coconut oil. J Biol Chem. 1955;215:15–26. - PubMed
    1. Korn ED. Clearing factor, a heparin-activated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart. J Biol Chem. 1955;215:1–14. - PubMed
    1. Havel RJ. Triglyceride-rich lipoproteins and plasma lipid transport. Arterioscler Thromb Vasc Biol. 2010;30:9–19. - PubMed
    1. Khera AV, et al. Myocardial Infarction Genetics Consortium; DiscovEHR Study Group; CARDIoGRAM Exome Consortium; Global Lipids Genetics Consortium Association of rare and common variation in the lipoprotein lipase gene with coronary artery disease. JAMA. 2017;317:937–946. - PMC - PubMed
    1. Stitziel NO, et al. Myocardial Infarction Genetics and CARDIoGRAM Exome Consortia Investigators Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease. N Engl J Med. 2016;374:1134–1144. - PMC - PubMed

Publication types

Associated data