Structure and mechanism of human diacylglycerol O-acyltransferase-1
EMSL Project ID
51365
Abstract
Diacylglycerol O-acyltransferase-1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in mammals. DGAT1 belongs to the superfamily of membrane-bound O-acyltransferases (MBOAT) that are found in all kingdoms of life and involved in lipid biosynthesis and remodeling and in protein acylation. However, the lack of 3-dimensional structures has limited our understanding of substrate recognition and mechanism of catalysis in DGAT1 and in members of the MBOAT superfamily, and hampers rational targeting of human DGAT1 (hDGAT1) for therapeutic purposes. We have expressed and purified hDGAT1 and demonstrated that the purified protein is enzymatically active. We have also optimized conditions for preparing cyo-EM grids and were able to obtain an initial structure at 3.1 Å resolution using cryo-EM single-particle reconstitution. These exciting new results established feasibility of a structure based approach to understand the enzymatic mechanism of hDGAT1. We propose to solve the following three structures by utilizing the PNCC Krios for cryo-EM. Aim 1. Solve the structure of hDGAT1 in complex with both of its substrates, diacylglycerol and oleoyl-coA. The structure will help us understand how the two substrates are recognized, how the active site is arranged, and how the catalysis may happen.
Aim 2. Solve the structure of hDGAT1 with its N-terminus deleted. The N-terminus of the hDGAT1 is known to regulate the enzymatic activity. The initial hDGAT1 structure shows that the N-terminal residues 64-83 interact with the neighboring protomer. We found that when these residues are deleted hDGAT1 lost its ability to bind oleoyl-CoA, indicating a structural change at the binding site. The structure of hDAGT1 with its N-terminus deleted will define conformational changes induced by binding of the N-terminus.
Aim 3. Solve the structure of hDGAT1 in its tetrameric state. The initial structure of hDGAT1 is in a dimeric form. However, biochemical studies showed that hDGAT1 can form both dimers and tetramers in cell membrane. We have developed a purification protocol to preserve the tetrameric form of hDGAT1, and we will solve the structure of the tetramer.
Project Details
Start Date
2020-05-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members