Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

Abstract

Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his-tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally occurring fluorescent cis-parinaroyl-CoA with very high affinity (K(d)=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his-tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor-4alpha (HNF-4alpha), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4alpha were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4alpha (intermolecular distance of 73 A) at high affinity (K(d)=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.

Figure 1. Mouse recombinant his-ACBP cloning and purification

A. Agarose gels of the his-ACBP PCR product, his-ACBP PCR fragment cloned into pGEM-T vector, and his-ACBP subcloned into pET21b vector: HMW, high molecular weight marker (1kb); LMW, low molecular weight marker (100bp); H-ACBP-PCR, PCR product encoding his-ACBP cDNA; pGEM-H-ACBP, his-ACBP PCR fragment T-A cloned into the pGEM-T plasmid: 1, 1μg of uncut plasmid; 2, 1μg of Xho I/Nde I cut plasmid; pET21b-H-ACBP refers to his-ACBP cDNA cloned into pET21b plasmid: 3, 1μg of Xho I -cut plasmid; 4, 1μg of Xho I/Nde I digested plasmid. B. DNA sequence of mouse ACBP cDNA inserted into pET21b vector and its amino acid translation. C. SDS-PAGE gel and Western blotting of his-ACBP expressed in BL21 E. coli cells in the absence (lanes 1 and 3, respectively) and presence of IPTG (lanes 2 and 4, respectively). For SDS-PAGE and Coomassie staining, 20 μg of total protein was loaded per lane, while 10 μg of total protein per lane was used for Western blotting. D. Representative Coomassie stained SDS-PAGE gel showing his-ACBP in E. coli cell lysate (lane 1, 25 μg protein) and elution fraction after Ni-column (lane 2, 2 μg protein). SDS-PAGE gel of purified his-ACBP silver stained to determine purity: 3, protein marker; 4, ultra low range protein marker; 5, 1μg of his-ACBP; 6, 2μg of his-ACBP; 7, 5μg of his-ACBP.

Figure 2. Structural characteristics of his-ACBP vs untagged ACBP: UV absorbance, CD and fluorescence spectra

A. UV-Vis spectra of 5μM his-ACBP (solid) and 5μM untagged ACBP (dashed). B. Far UV CD spectra of his-tagged ACBP (solid) and ACBP (open). All CD studies were performed with 4μM protein. C. Fluorescence emission spectra of 200nM his-ACBP (solid) and 200nM ACBP (dashed) with excitation at 280 nm. D. Fluorescence emission spectra of 200nM his-ACBP (solid) and 200nM ACBP (dashed) with excitation at 295 nm.

Figure 3. Affinity and number of binding sites of his-ACBP with cis-parinaroyl-CoA (cPNCoA) and cis-parinaric acid (cPNA)

Scatter plot and curve fitting of maximal fluorescence intensity (ex 310 nm, em 410 nm) with increasing of cPNCoA (A) and cPNA (A inset) in presence of 30nM his-ACBP. Reverse titrations of constant amounts (50nM) of cPNCoA (B) and cPNA (B inset) with increasing concentrations of his-ACBP indicated saturation at a molar ratio of 1 with cPNCoA, and nonspecific binding for cPNA. C. UV spectra of cPNCoA (4 μM) in potassium phosphate buffer pH 7.4 (solid), cPNCoA spectra with increasing his-ACBP (0.4-16 μM) are represented by dashed and dotted lines. D. Ratios of absorbance of valley 1 (V1)/peak 2 (P2) from spectra in C plotted vs protein/ligand molar ratio. E. Wavelengths of peak 2 (P2) in C plotted vs protein/ligand molar ratio.

Figure 4. Structural and functional characterization of Cy5-labeled his-ACBP

A. SDS-PAGE of Cy5-his-ACBP and his-ACBP. B. Western blotting of Cy5-his-ACBP and his-ACBP, 500ng of protein per lane. C. Far UV CD spectrum of his-ACBP and Cy5-his-ACBP. All CD studies were performed with 4μM protein. D. Titration and binding curve of Cy5-his-ACBP with cis-parinaroyl-CoA.

Figure 5. Interaction of his-ACBP with HNF-4α by FRET

Cy3-HNF-4α (200 nM) and Cy3-β-galactosidase (200 nM, negative control) were titrated with increasing Cy5-his-ACBP (5 nM to 2 μM) as described in Methods. A. Emission spectra of Cy3-HNF-4α/Cy5-his-ACBP with donor Cy3 excitation at 500 nm; inset, quenching of donor Cy3 fluorescence vs acceptor Cy5-his-ACBP concentrations. B. Close-up of spectra in A, 640-750 nm range; inset, sensitized emission intensity for acceptor Cy5-his-ACBP vs Cy5-his-ACBP concentration. C. Emission spectra of Cy3-β-galactosidase excited at 500 nm; inset, quenching in Cy3 versus Cy5-his-ACBP concentrations.