ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis

Abstract

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.

Keywords: ATP-citrate lyase; acetyl coenzyme A (acetyl-CoA); citrate synthase; coenzyme A (CoA); enzyme mechanism; metabolism; protein assembly.

Figure 1.

Production of recombinant human ACLY and truncations. A, construct map of ACLY. Domains 1–5 are numbered according to domain arrangement in bacterial SCS. B, SDS-PAGE of ACLY constructs on 12% Tris-MOPS. C, single representative SEC traces of ACLY constructs on Superose 6 increase chromatography column. The protein concentrations at the time of loading the column were 40 μm for ACLY-FL, 100 μm for ACLY-Nterm, and 40 μm for ACLY-Cterm. MW Std, molecular weight standard; C-term, C-terminal; N-term, N-terminal.

Figure 2.

Sedimentation equilibrium of ACLY truncations. Natural log (LN) linearized radial distribution of SE data obtained through Absorbance_{280 nm} optics for samples at 0.4 mg ml⁻¹, centrifuged for 24 h at 9000 rpm. The experimental data (purple) were plotted over the square distance of the center of rotation. These were compared with simulated monomer (gray) to tetramer (dark blue) distributions for ideal globular proteins corresponding to multiples of the monomer molar mass of the corresponding ACLY species. A, ACLY-FL. B, ACLY-Nterm. C, ACLY-Cterm.

Figure 3.

Determination of multimeric state equilibria of the ACLY constructs in solution. A, intensity percentage distribution of hydrodynamic radii (Rh) measured by dynamic light scattering of ACLY full-length and truncation constructs at 8 μm and 3.3 °C across two independent experiments, n = 6 replicates each. B, sedimentation velocity of 0.3 mg ml⁻¹ protein at 42,000 rpm and 4 °C across two independent experiments.

Figure 4.

Western blotting analysis of ectopic ACLY-FL co-IP in HEK293T cells. Immunoprecipitation of ACLY-FL with N-terminal Myc epitope, tested with anti-FLAG for evidence of association, with detection by anti-Myc as confirmation of pulldown is shown. Ectopic expression was confirmed through blotting of the pulldown inputs. The levels of β-actin in the input were used a loading control. IB, immunoblot.

Figure 5.

Differential scanning calorimetry of ACLY full-length and truncations at 25 μm. Scans were run from 10 to 90 °C at a 60 °C h⁻¹ ramp rate. Representative traces of independent experiments are shown, with the average T_M of all experiments reported above each unfolding transition event.

Figure 6.

Co-migration of ACLY-Nterm and ACLY-Cterm. A, SEC of ACLY-Nterm (5 μm) and ACLY-Cterm (20 μm) individually or after 1-h co-incubation on a Superdex 200 increase chromatography column. B, SDS-PAGE of SEC eluate for each SEC run on 12% Tris-MOPS. MW, molecular mass; MW Std, molecular mass standard; C-term, C-terminal; N-term, N-terminal.

Figure 7.

MDH-coupled enzyme activity assay of ACLY constructs at 20 nm each, as well as titration of ACLY-Cterm at varying concentrations into ACLY-Nterm at 20 nm. All experiments were carried out under cell-free conditions with three independent experiments, n = 6 replicates each. n.s., not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 8.

Differential scanning fluorimetry assay for ACLY affinity for substrates. Each construct was analyzed at 0.1 mg ml⁻¹ with saturating concentrations of citrate, ATP, MgCl₂, and/or CoA. Summary of two independent experiments is shown, n = 6 replicates each. Statistical significance was calculated by Dunnett's multiple comparisons test. n.s., not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 9.

Differential scanning calorimetry of ACLY full-length and truncations at 25 μm with or without CoA at 500 μm. Representative traces of independent experiments are shown, with average T_M overlaid. Scans were run from 10 to 90 °C at a 60 °C hr⁻¹ ramp rate. n.s., not significant; * p ≤ 0.05.