Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT–HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

(a) 2D class averages generated from ISAC with 178 classes accounting for 8,507 particle projections. The number of particles in each class is shown in the bottom left and the class number in the bottom right. (b) Representative 2D class averages from Relion showing that up to five LCATs can bind to a single HDL particle. This heterogeneity helps to explain XL-MS results that are inconsistent with the most abundant assembly. (i) Isolated HDL particle (710 particles). (ii) One LCAT bound (555). (iii) Side/edge view of one LCAT bound (233) showing that it binds in a manner that is staggered with respect to the Apo-AI belt, as suggested by our 3D reconstructions (see Fig. 4a). (iv) Two LCAT bound with roughly 60˚ angular separation around the HDL disc (470). (v) Two LCATs bound, but this time with about 120˚ of separation (270). (vi) A side view of two LCAT bound with 120˚ of separation (233). (vii) Three LCATs bound (162), indicating that the enzyme has a propensity to bind at roughly 60˚ intervals around the circumference of the HDL. (viii) Five LCATs bound (66). The underlying message is that although typically only one LCAT would be expected to bind to an HDL in vivo and although there may be preferential binding to a particular Apo-AI region, the binding of LCAT to HDL is also somewhat nonspecific, indicating that context (amphipathic helices at the edge of the HDL lipid bilayer) is more important than sequence. However, activity may be highly dependent on sequence or the region of ApoA-I that is bound. (c) Angular distribution for the 2:1 LCAT:HDL 3D map showing the preferred orientation for the view normal to the plane of the HDL disc. Each cylinder represents an angular viewpoint, with the tallest red cylinder equal to the highest number of particles in that orientation.
Supplementary Fig. 2: The angular separation for two bound LCAT monomers is consistent with binding to helix 6 region of ApoA-I.
(a) The angle between the two LCATs in 2D class averages was measured using ImageJ 1 . The angles from 51 classes were then plotted as a histogram, with the number of particles in each class used to show the distribution in the dataset. Representative class averages are shown above the plot. Below, the arrows point to two models for how LCAT could bind to the ApoA-I belt to create the observed range of angles, with each binding at helix 6 (H6) or more towards H7. The ApoA-I monomers are each colored as a rainbow from H1 to H10 in the double belt model 2 . (b) The 3D reconstruction shows that two LCAT molecules bind to the side of the disc with an offset of ~15 Å along the central axis of the disc (left), which is consistent with them being bound to distinct ApoA-I chains, which are spaced ~12 Å apart in the double belt model (right). (c) The different registers of the ApoA-I double belt as proposed by the Davidson lab 3 would result in different binding modes for LCAT, here shown for example with LCAT bound to helix 6. 5/5 indicates H5 aligned with H5, etc. with the top showing the side view and the bottom image showing how LCAT would orient in a top-down view. The distribution observed in a is not consistent with alternative models presuming that LCAT preferentially binds to a specific helix (not necessarily H6). (a) 4-20% SDS-PAGE showing individual fractions from the crosslinked LCAT-HDL chromatogram (below). Fractions 6-7 were analyzed together (referred to as peak 1), and fraction 8 separately (referred to as peak 2). (b) Map of crosslinks identified within (purple) and between (green) LCAT and ApoA-I. The dashed lines refer to ambiguous crosslinks that occur within isolated peptides, such as the ambiguity between LCAT Lys105 and Ser108 (although Lys105 is more reactive). Supplementary Fig. 4: Mass spectrometry workflow.
Crosslinks were identified using a mass spectrometry workflow that employed two separate programs: MeroX 2.0, which identifies crosslinks at the MS 2 level, and the XlinkX node in Proteome Discoverer 2.2, which identifies pairs at the MS 2 level and fragmentation at the MS 3 level. The amino acid sequence for human ApoA-I is shown at the top of each line. Dark blue bars represent peptic peptides identified and followed by HDX-MS for both ApoA-I in HDL alone and in HDL bound to LCAT. Supplementary Fig. 10: Relative deuterium uptake curves for all peptides in the sequence coverage map. Supplementary Fig. 10 (cont) Free LCAT deuterium uptake (red) is compared to LCAT within the LCAT-HDL complex (blue) at 5 time-points, followed by free ApoA-I within HDL (black) compared to ApoA-I within the LCAT-HDL complex. Supplementary Fig. 11: ApoA-I HDX data mapped onto the double belt HDL model.
(a) The relative deuterium uptake for free HDL following ApoA-I peptides identified by HDX-MS, whereas (b) shows the difference (Dcomplex -DHDL) between relative deuterium exchange in HDL when free and bound to LCAT. In both the exchange time is indicated at the top of the panel, increasing from 10 s to 30 min from left to right. The panels are color-coded according to the scales at the bottom, with free HDL uptake in increasing percentages (a, c) and the differences shown in Da (b, d). Down the left side are the residue numbers of each peptide fragment, arranged from Nto C-terminus (top to bottom). Peptides highlighted with red numbering are representative of the deuterium incorporation and were plotted onto the double belt model 2 in (c) for free HDL at the 3 min timepoint and (d) for the differences between free and LCAT-bound at the 10 s timepoint. Supplementary Fig. 12: HDX data mapped onto open and closed LCAT structures. (a) The first panel shows the relative deuterium uptake for free LCAT for all peptides identified and followed by HDX-MS. The exchange time is indicated at the bottom of the panel, increasing from 10 s to 30 min from left to right. All differences are shown in Da and are color-coded according to the scale at the bottom. Down the left side are the residue numbers of each peptide fragment, arranged from N-to C-terminus (top to bottom). Peptides highlighted with red numbering are representative of the differences in relative deuterium incorporation and were plotted onto two x-ray crystal structures of LCAT, (b) an open LCAT structure (PDB entry 6MVD) and (c) a closed lid conformation (PDB entry 5TXF) for the 10 min time point using PyMOL software.  K240  K140/S142  239-LKEEQR-244  137-LQEKLSPLGEEMR-149  263  148  27  8  3  213  133  6  2  2   K105/S108  K140/S142  100-VPGFGKTYSVE-110  137-LQEKLSPLGEEMR-149  203  91  16  8  3  206  35  1  2