A retractable lid in lecithin:cholesterol acyltransferase provides a structural mechanism for activation by apolipoprotein A-I

Abstract

Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.

Interaction of lecithin-cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I derived peptides: implications for the cofactor mechanism of apolipoprotein A-I

“…The simulation results showed that the lid-region of LCAT forms a coil secondary structure accompanied with relatively high residual fluctuations when compared to the other structural parts of LCAT. The fact that the lid regions are partly missing from the X-ray structures of LCAT is backing our simulation results showing a highly mobile region with no definite structural conformation (Ruwanthi N. Manthei et al, 2017; Derek E. . Another important finding was that the highly dynamical lid-loop shields the non-polar amino acids located at the tunnel opening from the water.…”

“…In this fashion, we strived to establish mechanistic insights to the conformational switching of the lid-loop that, presumably, is a prerequisite for the esterification reaction of LCAT to take place. Moreover, the overall stability of the LCAT structure was assessed against the X-ray structures to validate our computational models (Ruwanthi N. Manthei et al, 2017;Derek E Piper et al, 2015). To construct applicable LCAT models for these purposes, the lid loop region (aa 233-247) was computationally grafted to the high resolution X-ray structures of LCAT representing either the open or closed state (Ruwanthi N. Derek E. Piper et al, 2015).…”

“…For years, investigations concerning the LCAT lipid bilayer interaction and activation by apoA-I have been hindered by a lack of detailed atomistic structure of LCAT. Recently, however, Xray structures of LCAT have become available enabling for example the computational studies of LCAT interacting with lipid surfaces and apoA-I to elucidate the mechanistic details concerning the cholesterol esterification process (Glukhova et al, 2015;Ruwanthi N Gunawardane et al, 2016;Manthei et al, 2017;Derek E. Piper et al, 2015). From the structures it is evident that the tertiary and secondary structure of LCAT is homological to lysosomal phospholipase A2 as pointed out by three similar folds: the cap-domain, the membrane-binding domain, and the a/b hydrolase domain (see Fig 1A) (Glukhova et al, 2015).…”

LCAT Enzyme Replacement Therapy Reduces LpX and Improves Kidney Function in a Mouse Model of Familial LCAT Deficiency

“…rhLCAT Treatment of Mice. rhLCAT (MEDI6012) was produced as previously described (Manthei et al, 2017). Human and mouse LCAT enzymes possess a high degree of homology (Peelman et al, 1999), and indeed we found 87% identity between human and mouse LCAT reference protein sequences by BLAST analysis.…”

Interleukin-32 upregulates the expression of ABCA1 and ABCG1 resulting in reduced intracellular lipid concentrations in primary human hepatocytes

“…A critical participant herein is the ATP-binding cassette A1 (ABCA1) expressed on liver cells and enterocytes. These nascent, discoidal apoA-I containing HDL particles are secreted via hepatic and enterocyte ABCA1 and are matured in circulation via the lecithine-cholesterolacyltransferase (LCAT) function and formation of spherical HDL particles [24]. In the absence of ABCA1, extremely low levels of HDLc and apoA-I are observed, which contribute to an increased risk for CVD.…”