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

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          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.

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          Author and article information

          Journal
          J Biol Chem
          J. Biol. Chem
          jbc
          jbc
          JBC
          The Journal of Biological Chemistry
          American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
          0021-9258
          1083-351X
          8 December 2017
          13 October 2017
          : 292
          : 49
          : 20313-20327
          Affiliations
          From the []Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109,
          [§ ]MedImmune, Gaithersburg, Maryland 20878,
          the []Department of Pharmaceutical Sciences and Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109, and
          the []Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109
          Author notes
          [2 ] To whom correspondence should be addressed: Dept. of Biological Sciences, 240 S. Martin Jischke Dr., Purdue University, West Lafayette, IN 47907-2054. Tel.: 765-494-180; Fax: 734-763-6492; E-mail: jtesmer@ 123456purdue.edu .
          [1]

          Present address: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences Biology, and Dept. of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.

          Edited by John M. Denu

          Article
          PMC5724016 PMC5724016 5724016 M117.802736
          10.1074/jbc.M117.802736
          5724016
          29030428
          f965cb3a-2618-4a3d-bf86-d5bf7024e90b
          © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
          History
          : 17 June 2017
          : 15 September 2017
          Funding
          Funded by: National Institutes of Health , open-funder-registry 10.13039/100000002;
          Award ID: HL071818
          Award ID: HL122416
          Award ID: AR056991
          Award ID: F32HL131288
          Funded by: American Heart Association , open-funder-registry 10.13039/100000968;
          Award ID: 15POST24870001
          Award ID: 16POST27760002
          Award ID: 13SDG17230049
          Funded by: U.S. Department of Veterans Affairs , open-funder-registry 10.13039/100000738;
          Award ID: 1I01BX002021
          Categories
          Protein Structure and Folding

          lecithin,HDX MS,structural biology,inhibitor,high-density lipoprotein (HDL),crystallography,conformational change,cholesterol,apolipoprotein,acyltransferase

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