Identification of the Citrate-binding Site of Human ATP-Citrate Lyase Using X-ray Crystallography

Tianjun Sun,Koto Hayakawa,Katherine S Bateman,Marie E Fraser

doi:10.1074/jbc.m109.078667

Abstract

ATP-citrate lyase (ACLY) catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP. In humans, ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. In situ proteolysis of full-length human ACLY gave crystals of a truncated form, revealing the conformations of residues 2-425, 487-750, and 767-820 of the 1101-amino acid protein. Residues 2-425 form three domains homologous to the beta-subunit of succinyl-CoA synthetase (SCS), while residues 487-820 form two domains homologous to the alpha-subunit of SCS. The crystals were grown in the presence of tartrate or the substrate, citrate, and the structure revealed the citrate-binding site. A loop formed by residues 343-348 interacts via specific hydrogen bonds with the hydroxyl and carboxyl groups on the prochiral center of citrate. Arg-379 forms a salt bridge with the pro-R carboxylate of citrate. The pro-S carboxylate is free to react, providing insight into the stereospecificity of ACLY. Because this is the first structure of any member of the acyl-CoA synthetase (NDP-forming) superfamily in complex with its organic acid substrate, locating the citrate-binding site is significant for understanding the catalytic mechanism of each member, including the prototype SCS. Comparison of the CoA-binding site of SCSs with the similar structure in ACLY showed that ACLY possesses a different CoA-binding site. Comparisons of the nucleotide-binding site of SCSs with the similar structure in ACLY indicates that this is the ATP-binding site of ACLY.

Highlights

IntroductionLike succinyl-CoA synthetase (SCS), ATP-citrate lyase (ACLY) is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) [5, 6]
Our understanding of the reaction mechanism of ATP-citrate lyase (ACLY) has been based on studies of this enzyme and of two enzymes with sequence similarity to ACLY
Peptide mass fingerprinting identified 10 peptides in the 50-kDa band and 12 in the 35-kDa band that matched peptides predicted to be cleaved from human ACLY (hACLY), confirming that the crystals contained truncated hACLY

Summary

Introduction

Like SCS, ACLY is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) [5, 6]. In the last step (Reaction 4), citryl-CoA is cleaved to give acetyl-CoA and oxaloacetate, the reverse reaction to that catalyzed by citrate synthase. The domains are numbered according to their order in Escherichia coli SCS, because this enzyme was the first member of the superfamily to have its structure determined using x-ray crystallography [9]. The C-terminal portion of ACLY shows sequence similarity to the large domain of citrate synthase [19]. As a step in obtaining the crystal structure of full-length ACLY, we crystallized a truncated form that contains residues from domains 1–5. The crystals were grown in the presence of tartrate or citrate and reveal both the structure of two-thirds of ACLY and the binding site of the substrate citrate

Methods

Results

Discussion

Conclusion