Fig. 2: LCAT–HDL crosslinking implicates preferential LCAT–HDL interactions.

a XL-MS workflow, see Methods for further details. PD refers to the XLinkX node of Proteome Discoverer. b SDS-PAGE gel of LCAT–HDL crosslinking experiments. Each lane is described above, with the protein(s) in that experiment, and an x specifying an experiment that included DC4 crosslinker. The xLCAT–HDL lane reveals new higher molecular weight species corresponding to crosslinked products. c SEC results for the crosslinked complex, compared to LCAT or HDL alone. The two shaded peaks were then subjected to MS and peptide identification, with results in Table 1. d, e Crosslinked LCAT residues are shown in red spheres on the d open (PDB code 6MVD) and e closed (PDB code 5TXF) LCAT crystal structures. Lys240 is shown with a nearby residue as it is disordered in all crystal structures. The dynamic LCAT lid is a darker gray to highlight the movement between the two structures and the Ser181 side chain in gray spheres as a marker for the active site. N-linked NAG sugars are shown with purple spheres and are expected to be excluded from the LCAT–HDL interface. f ApoA-I crosslinks are mapped on the ApoA-I primary sequence with two proteins depicting the double belt orientation as shown below and to the right¹⁸. Crosslinks are shown with a red x on the sequence and red spheres in the structures. ApoA-I helices (H1–10) are colored the same in both subunits. The yellow N-terminal region of ApoA-I is not included in the double belt model. The shaded boxes indicate the hypothesized LCAT-binding location. Lipids are shown as gray sticks in the middle of the ApoA-I belt in each structure.