Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site

Fig. 1. LPL forms C-terminal to C-terminal homodimers. a The 2D class of LPL’s preferred orientation at 0° (left). Tilting the grid to 45° allowed us to capture an alternate view of this orientation (right). b The density map for the LPL dimer (purple). The dimerization interface is between the C-terminal domains of both LPL subunits. Density corresponding to the glycosylation of N73 was observed. c When thresholding of the density map is increased, density for the lid domain of LPL appears (aqua). The tryptophan loop in the C-terminal domain, which is involved in substrate recognition, can be seen along the same plane as the density corresponding to the lid peptide (cyan). The lid peptide is likely too mobile to be resolved at high resolution. The lid domain density has shifted downward and to the side of the active site (orange) adopting an open conformation. d The resolution of the LPL dimer is 3.9 Å with a sphericity of 0.85 out of 1, which indicates that we were able to create a nearly complete reconstruction, despite anisotropy resulting from the preferred orientation of the particles (Supplementary Fig. 3B). Source data are provided as a Source Data file. e The molecular map of…

Fig. 2. The LPL homodimer reveals an open active site pore. a The LPL dimer map colored to show the LPL active site residues (orange) and the tryptophan loop (cyan), which is involved in substrate recognition. An area without protein density is seen in the N-terminal of LPL suggested the existence of a pore spanning the protein. b We analyzed the LPL model and were able to identify a pore (green) that spanned the N-terminal domain, shown here in the density map. The pore is located adjacent to the active site and an aqua arrow indicates the position of the lid density if thresholding were lower to the side of the pore. A cutaway of the density shows that the pore spans the N-terminal domain of LPL. c MOLEonline was used to create a trace of the characteristics of the amino acids that line the identified pore. The residues corresponding to the entrance to the LPL pore are at the top of the diagram and the path of the pore travels to the terminus at the bottom. The pore is 44.5 Å in length with a bottleneck radius of 1.4 Å. The location of the active site is marked by a dotted orange line. Charge and hydropathy colors correspond to the legends above the diagram. The source data for this…

Fig. 3. A pore is not present in other open lipase structures. a A transparent surface representation of a single subunit of LPL (purple) from the LPL dimer, with the path of the pore (lime green). b A cutaway shows the path of the pore through the protein. c The density corresponding to the LPL lid domain (blue) has swung to the side giving access to the active site and pore. d A closeup look at the LPL pore cutaway, with the active site residues (red) and other residues colored on the surface to show hydrophobic (yellow) and hydrophilic (aqua) character. The cutaway surface is in purple. e Alignment of LPL and two pancreatic triacylglyceride lipase (PTL) structures (cyan (1LPA) and green (1ETH)) shows similar folds and well aligned location of the active site residues (red). f A cutaway of the structure of human PTL (cyan) aligned to LPL – no pore is observed, only a small pocket adjacent to the active site (red). The PTL/procolipase complex was crystallized in the presence of phophatidyl choline micelles and bile salt (PDB 1LPA). A zoom-in of the active site in the cross-section shows a diundecyl phosphatidyl choline (PLC) bound (lime green). g PTL in the presence of procolipase was…

Fig. 4. The LPL dimerization interface overlaps with other known interfaces, including LPL/GPIHBP1 and the LPL helix. The structure of LPL (purple) has been solved in 3 distinct states. a An active homodimer described in this work (one LPL subunit is colored lime green), b a heterodimer with GPIHBP1 (gold) (shown as PDB 6OB0), and c an inactive helix (EMDB 20673), for which the repeating subunits are inactive LPL dimers (purple and magenta, the rest of the LPL in the helix is colored gray). We used PDBePISA to identify the interacting residues for each of these complexes. This analysis is available as Supplementary Data 9. d In the LPL homodimer the interaction interface was located at the C-terminal, and we mapped the residues identified as being buried by the interface onto a space filling representation of one monomer of the LPL homodimer (interface residues, colored lime green). e The LPL residues interfacing with GPIHBP1 (e, gold) are mapped onto the same LPL, as are the helical interfaces (f, gray) and the inactive dimer interface (g, magenta). The helical interfaces represent the interactions that occur in the LPL helix that are not with the companion LPL subunit found in the…

Fig. 5. Concentration Drives LPL oligomerization. a Mass photometry of LPL (blue) at increasing concentrations shows that when LPL is analyzed at low concentrations it forms a monomer, and as LPL concentration increases a higher molecular weight shoulder appears. At 100 nM LPL the primary peak corresponds to the molecular weight of an LPL dimer. b LPL in PBS with 0.25 mM deoxycholate (green) displays a similar trend to LPL without deoxycholate. c LPL measured in PBS with 27 U/mL heparin (purple) also shows the shift to dimerization with increasing concentration. However, the appearance of a dimeric molecular weight peak is observed at lower concentration (50 nM) compared to LPL or LPL with deoxycholate. The data was fitted with single or multiple Gaussian distributions to determine an average molecular weight (kDa) and the width of the standard deviation of the fitted Gaussian is given by sigma (σ). The combined trace of the multiple Gaussians is also shown to illustrate the fit of the Gaussians to the histograms. LPL monomer theoretical molecular weight = 50.5 kDa. Source data are provided as a Source Data file.

Fig. 6. Crosslinking LPL captures monomers, dimers, trimers, and tetramers. To capture and analyze LPL oligomers, we used gradient fixation (GraFix) to gently crosslink and separate protein by molecular weight. LPL alone (a) and with deoxycholate (b) showed an array of oligomers forming. The position of uncrosslinked LPL is marked in red. Blots were developed using the monoclonal LPL antibody 5D2, and the first observable LPL oligomer runs below the uncrosslinked LPL molecular weight—likely due to crosslinking altering its electrophoretic mobility. At least 4 distinct LPL oligomers are observed in both samples (black arrows). The similarity of LPL with and without deoxycholate suggests that the deoxycholate does not alter the overall oligomeric state of the protein. GraFix crosslinking experiments were performed in triplicate. The results of the gradient fractionations in (a) and (b) were analyzed using mass photometry to determine the exact molecular weight of their components. c LPL fraction 9 (blue) and d LPL with deoxycholate fraction 9 (green) are shown as representative data. The data was fitted with multiple Gaussian distributions to determine the average molecular weight (kDa)…

Fig. 7. Proposed Unidirectional Model for Triglyceride Hydrolysis by LPL. a Prior to associating with a substrate (gray), the lid peptide (dark blue) covers access to the LPL (purple) active site. Hydrophobic tryptophan residues (yellow) in the C-terminal domain of LPL are involved in substrate recognition41. b After association with a substrate, LPL undergoes interfacial activation, where the association with a nonpolar-aqueous interface facilitates the lid peptide swinging to the side. c Following lid-peptide opening, the presence of triglyceride (red/light blue) or a hydrophobic environment triggers formation of an acyl-chain binding pore (lime green). d The hydrophobic acyl chain (red) enters the pore positioned so that the lipase active site residues can hydrolyze the ester bond connecting the acyl chain to the glyceride backbone (light blue). e Following hydrolysis, the free fatty acid (FFA) exits the pore from the opposite side it entered, resulting in unidirectional FFA travel. The remaining ligand can continue to be hydrolyzed at other acyl chains or transfer back to the hydrophobic substrate. This unidirectional model efficiently allows FFA to transfer away from their original…

Fig. 8. Proposed model of LPL dimer formation in the capillary. LPL (purple) is synthesized in the adipose and oxidative tissues and secreted into the interstitial space, where it binds to GPIHBP1 (yellow) as a heterodimer8,9. a GPIHBP1 transports LPL across the endothelium (salmon) into the capillary (pink)7, where it interacts with triglyceride rich lipoproteins, including chylomicrons (gray) and VLDL10. b LPL hydrolyzes triglycerides to release free fatty acids which trigger dissociation from GPIHBP166—allowing LPL to form a homodimer, maintaining activity and protein stability. c Following continued triglyceride hydrolysis, the lipoprotein remnant is released from all associations that keep it tethered to the capillary wall and the LPL homodimers remain on the remnant11. d The lipoprotein remnant and attached LPL homodimer travels through the capillaries to the liver and pass into the space of Disse, where the remnant binds to hepatic transmembrane protein, LRP (green). LRP has previously been shown to interact with LPL homodimers to enhance uptake of low-density lipoprotein remnants into hepatocytes (lime green)67,68.