Structural basis for catalysis and substrate specificity of human ACAT1

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are key enzymes in cholesterol homeostasis, belonging to the membrane-bound O-acyltransferase (MBOAT) family.
• ACATs catalyze the formation of cholesteryl esters, essential for cellular cholesterol storage and transport.
• ACATs are implicated as therapeutic targets for atherosclerosis, Alzheimer's disease, and cancer.

Purpose of the Study:

• To determine the high-resolution structure of human ACAT1 using cryo-electron microscopy.
• To elucidate the structural basis for ACAT1's enzymatic activity and substrate specificity.
• To provide insights into the catalytic mechanism of ACAT1 and other MBOAT family enzymes.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of human ACAT1.
• Dimer of dimers quaternary structure analysis.
• Structure-guided mutational analyses to probe substrate entry pathways.

Main Results:

• The cryo-EM structure of human ACAT1 was resolved as a dimer of dimers.
• Each protomer comprises nine transmembrane segments, forming distinct cytosolic and transmembrane tunnels.
• These tunnels converge at the active site, suggesting separate entry routes for acyl-coenzyme A (cytosolic tunnel) and cholesterol (transmembrane tunnel).
• Biochemical data rationalize ACAT1's preference for unsaturated acyl chains.

Conclusions:

• The determined structure provides a molecular framework for understanding ACAT1 function.
• The identified substrate entry tunnels offer mechanistic insights into ACAT1 catalysis.
• This study advances our understanding of cholesterol metabolism and provides a basis for rational drug design targeting ACAT1.