Novel Molecular Interactions of Acylcarnitines and Fatty Acids with Myoglobin

Abstract

Previous research has indicated that long-chain fatty acids can bind myoglobin (Mb) in an oxygen-dependent manner. This suggests that oxy-Mb may play an important role in fuel delivery in Mb-rich muscle fibers (e.g. type I fibers and cardiomyocytes), and raises the possibility that Mb also serves as an acylcarnitine-binding protein. We report for the first time the putative interaction and affinity characteristics for different chain lengths of both fatty acids and acylcarnitines with oxy-Mb using molecular dynamic simulations and isothermal titration calorimetry experiments. We found that short- to medium-chain fatty acids or acylcarnitines (ranging from C2:0 to C10:0) fail to achieve a stable conformation with oxy-Mb. Furthermore, our results indicate that C12:0 is the minimum chain length essential for stable binding of either fatty acids or acylcarnitines with oxy-Mb. Importantly, the empirical lipid binding studies were consistent with structural modeling. These results reveal that: (i) the lipid binding affinity for oxy-Mb increases as the chain length increases (i.e. C12:0 to C18:1), (ii) the binding affinities of acylcarnitines are higher when compared with their respective fatty acid counterparts, and (iii) both fatty acids and acylcarnitines bind to oxy-Mb in 1:1 stoichiometry. Taken together, our results support a model in which oxy-Mb is a novel regulator of long-chain acylcarnitine and fatty acid pools in Mb-rich tissues. This has important implications for physiological fuel management during exercise, and relevance to pathophysiological conditions (e.g. fatty acid oxidation disorders and cardiac ischemia) where long-chain acylcarnitine accumulation is evident.

Keywords: isothermal titration calorimetry (ITC); lipid binding protein; lipotoxicity; molecular docking; molecular dynamics; myoglobin; polyunsaturated fatty acid (PUFA).

FIGURE 1.

Cluster analysis from AutoDock, displaying docking conformations of horse (A) deoxy-Mb with PLM, (B) oxy-Mb with PLM, (C) deoxy-Mb with PLC, and (D) oxy-Mb with PLC. The best 20 models for PLM and PLC are displayed as teal-blue lines and each representative set representative model is displayed as a stick. Similar patterns were observed for all the different chain lengths starting from C12 and up to C20, where the ligands for deoxy-Mb are docked close to the lysine residues (K56 and K50) and the ligands for oxy-Mb were placed near the porphyrin ring and the hydrophobic core close to the residues (K45 and K63).

FIGURE 2.

Snapshots of horse oxy-Mb with butyroylcarnitine (A–C) and palmitoylcarnitine (D–F) over time during MD simulations. A, 0 ns; B, 3 ns; and C, 6 ns are the time intervals for butyroylcarnitine; and D, 0 ns; E, 50 ns; and F, 100 ns are the time intervals for palmitoylcarnitine. The protein backbone is represented as ribbon shapes, whereas butyroylcarnitine (orange), palmitoylcarnitine (purple), Lys⁴⁵, Lys⁶³, and heme are displayed as sticks. Oxygen molecules are displayed as red ball shapes adjacent to the heme moiety. Butyroylcarnitine (circled) leaves the binding pocket rapidly, whereas palmitoylcarnitine remains stable in the hydrophobic pocket. Water molecules and ions are excluded for clarity.

FIGURE 3.

Structure of horse oxy-Mb interacting with fatty acids. A, laurate; B, myristate; C, palmitate; D, oleate; E, arachidonate and acylcarnitines; F, lauroylcarn; G, myristoylcarn; H, palmitoylcarn; I, oleoylcarn; J, arachidonoylcarn. Snapshots of only the fatty acid binding pocket are shown, with various ligands occupying the hydrophobic core near the porphyrin ring. The protein backbone is represented as a ribbon (colored pink), heme as sticks (colored gray), and ligands as sticks (colored cyan). All the ligands appear as the characteristic U-shaped structure, which warps around the oxygen molecule (colored red) with the exception of laurate (A) having linear and arachidonate (E) and arachidonoylcarn (J) attaining S-shaped structure.

FIGURE 4.

Histograms of percent of hydrogen-bond occupancies for interactions between different ligands with basic residues Lys⁴⁵ and Lys⁶³ of horse oxy-Mb, across 100-ns MD simulations.

FIGURE 5.

The averaged r.m.s. fluctuation of different fatty acids and acylcarnitines that stably bind oxy-Mb, as a function of time bound to oxy-Mb, showing the deviations by residue. Only heavy atoms (carbon, oxygen, and nitrogen) are taken into consideration, whereas calculating r.m.s. fluctuations (hydrogens were excluded). Total number of atoms in each ligand molecule; laurate (14), myristate (16), palmitate (18), oleate (20), arachidonate (22), lauroylcarnitine (24), myristoylcarnitine (26), palmitoylcarnitine (28), oleoylcarnitine (30), and arachidonoylcarnitine (32).

FIGURE 6.

Isothermograms representing the binding of lysozyme, Met-Mb, and oxy-Mb with different chain lengths of fatty acids and acylcarnitines. Within each metabolite isothermogram, the upper panel depicts the raw data of titration of reactants with time in minutes on the x axis and the energy releases/absorbed per second on the y axis. The lower panel is the integrated data with the molar ratio on the x axis and energy released/absorbed per injection on the y axis. The solid line in the bottom panels represents the best-fit of the experimental data, using a one-set of sites binding model from MicroCal Origin^TM.