Coleman Bethany, Bedi Shimpi, Hill John H, Morris Jamie, Manthei Kelly A, Hart Rachel C, He Yi, Shah Amy S, Jerome W Gray, Vaisar Tomas, Bornfeldt Karin E, Song Hyun, Segrest Jere P, Heinecke Jay W, Aller Stephen G, Tesmer John J G, Davidson W Sean
Department of Molecular and Cellular Biosciences, University of Cincinnati, Cincinnati, OH, USA.
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA.
J Lipid Res. 2025 May;66(5):100786. doi: 10.1016/j.jlr.2025.100786. Epub 2025 Mar 25.
Lecithin:cholesterol acyltransferase (LCAT) is a high-density lipoprotein (HDL) modifying protein that profoundly affects the composition and function of HDL subspecies. The cholesterol esterification activity of LCAT is dramatically increased by apolipoprotein A-I (APOA1) on HDL, but the mechanism remains unclear. Using site-directed mutagenesis, cross-linking, mass spectrometry, electron microscopy, protein engineering, and molecular docking, we identified two LCAT binding sites formed by helices 4 and 6 from two antiparallel APOA1 molecules in HDL. Although the reciprocating APOA1 "belts" form two ostensibly symmetrical binding locations, LCAT can adopt distinct orientations at each site, as shown by our 9.8 Å cryoEM envelope. In one case, LCAT membrane binding domains align with the APOA1 belts and, in the other, the HDL phospholipids. By introducing disulfide bonds between the APOA1 helical domains, we demonstrated that LCAT does not require helical separation during its reaction cycle. This indicates that LCAT, anchored to APOA1 belts, accesses substrates and deposits products through interactions with the planar lipid surface. This model of the LCAT/APOA1 interaction provides insights into how LCAT and possibly other HDL-modifying factors engage the APOA1 scaffold, offering potential strategies to enhance LCAT activity in individuals with genetic defects.
卵磷脂胆固醇酰基转移酶（LCAT）是一种高密度脂蛋白（HDL）修饰蛋白，它对HDL亚类的组成和功能有深远影响。HDL上的载脂蛋白A-I（APOA1）可显著提高LCAT的胆固醇酯化活性，但其机制尚不清楚。我们运用定点诱变、交联、质谱分析、电子显微镜、蛋白质工程和分子对接技术，在HDL中确定了由两个反平行APOA1分子的螺旋4和螺旋6形成的两个LCAT结合位点。尽管往复运动的APOA1“带”形成了两个表面上对称的结合位置，但如我们9.8 Å的冷冻电镜包膜所示，LCAT在每个位点可采取不同的取向。在一种情况下，LCAT膜结合结构域与APOA1带对齐，而在另一种情况下，与HDL磷脂对齐。通过在APOA1螺旋结构域之间引入二硫键，我们证明LCAT在其反应周期中不需要螺旋分离。这表明，锚定在APOA1带上的LCAT通过与平面脂质表面的相互作用来接触底物并沉积产物。这种LCAT/APOA1相互作用模型为LCAT以及可能其他HDL修饰因子如何与APOA1支架结合提供了见解，为增强有基因缺陷个体的LCAT活性提供了潜在策略。
