Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein

Abstract

Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2-20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

Keywords: CMC (critical micellar concentration); ITC (isothermal titration calorimetry); rCPT-2 (rat carnitine-palmitoyltransferase); β-OG (n-octyl-β-D-glucopyranoside).

Graphical abstract

Fig. 1

Critical micellar concentration (CMC) of β-octyl glucoside (β-OG) in the presence of DMSO and rCPT-2 inhibitors 1–4. (A) Influence of DMSO on CMC of β-OG in absence of inhibitors. (♦) β-OG without DMSO, (▴) β-OG + 1% (v/v) DMSO, (•) β-OG + 7.5% (v/v) DMSO. (B) Influence of 7.5% (v/v) DMSO on CMC of β-OG in presence of inhibitors. The inhibitor concentration at the CMC varies between ca. 70–100 μM. (•) β-OG only, (▴) β-OG + inhibitor 1, (▾) β-OG + inhibitor 2, (★) β-OG + inhibitor 3, (♦) β-OG + inhibitor 4. (C) Enthalpy of micelle formation as a function of temperature. (•) β-OG, (▴) β-OG + inhibitor 1, (▾) β-OG + inhibitor 2 (★) β-OG + inhibitor 3, (♦) β-OG + inhibitor 4.

Fig. 2

Calorimetric titration of rCPT-2 with inhibitor 1 at 10 °C. The inhibitor (140 μM) was injected with 25 steps of 10 μl into the calorimetric sample cell containing 11 μM of rCPT-2. (A) Heat flow as a function of time. (B) Reaction enthalpy, δh_i, of inhibitor 1 versus injection number. The solid line corresponds to the theoretical model assuming a 1:1 binding stoichiometry, a reaction enthalpy of ΔH⁰ = −4.7 kcal/mol and a binding constant of K = 8 × 10⁵ M⁻¹. Buffer composition: 25 mM Tris/HCl pH 8, 150 mM NaCl, 2 mM TCEP, 1% (w/v) β-OG, 1% (v/v) DMSO.

Fig. 3

Structures of rCPT-2 with bound inhibitors 1–4 as determined by X-ray crystallography and computational modeling. (A) Superposition of crystallographic and modeled structures of complexes of rCPT-2 with inhibitors 1 and 2 (blue and yellow, respectively, both modeled based on the crystal structure of a related piperidine class inhibitor, see Supplementary Data), the two isosteric surrogates of inhibitor 3 (cyan and green) and inhibitor 4 (ST1326, magenta). The solvent accessible surface of the carboxy-terminal domain of rCPT-2 (off-white, L441-I656) is shown with hydrophobic residues depicted in green and polar residues shown in orange. The amino terminal domain was removed for clarity. (B) Detail of the complex structure of rCPT-2 (green) with a surrogate of inhibitor 3 (cyan) that shows the interaction of the benzoic acid head group of this inhibitor class with the catalytic residue His372. The minimum distance is 2.8 Å. The final 2FoFc electron density of the structure is shown as gray mesh. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Proton transfer upon binding of inhibitor 3. The measured binding enthalpy for the interaction of inhibitor 3 with rCPT-2, ΔH_obs, is plotted versus the ionization enthalpies of different buffers. Measurements were made in HEPES/NaOH (ΔH_diss = 3.9 kcal/mol), Bicine/NaOH (ΔH_diss = 6.3 kcal/mol) and Tris/HCl (ΔH_diss = 11.5 kcal/mol) at pH 8 and 10 °C. The solid line is the linear regression analysis of the data.

Fig. 5

Experimental DSC scans on rCPT-2 with and without inhibitor 4. Black line: rCPT-2, no inhibitor, Red line: rCPT-2 in the presence of inhibitor 4. rCPT-2 concentration 14 μM, inhibitor 4 concentration 100 μM; buffer conditions as in Fig. 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)