An environment-dependent structural switch underlies the regulation of carnitine palmitoyltransferase 1A

Abstract

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid β-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nβ, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nβ to be noninhibitory, with the relative Nα/Nβ ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nβ ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

FIGURE 1.

Overview of CPT1A system. A, domain organization and homology model of the CPT1A structure without the N-terminal regulatory element (N) for which no homologous structure exists. Possible N association states (OMM-associated, free in cytosol, and CD-associated) are shown schematically. Bound MCoA is illustrated in ball-and-stick representation. The CPT1A membrane topology is known (60), but the relative orientations of the transmembrane (TM) and CD domains are unknown, and depicted is one possible arrangement. B, chemical structures of physiological enzyme inhibitor (MCoA) and substrates (palmitoyl-CoA (PCoA) and carnitine). The common CoA moiety of inhibitor and substrate is noted. C, sequence alignment of N of CPT1A and -1B. Conserved amino acids are colored by the Jalview multiple alignment editor (61) using the ClustalX color scheme.

FIGURE 2.

Comparison of employed micelles systems and ensuing NMR spectra. A, chemical structures of successfully employed detergents DAC, DDAC, TDAC, and HDAC, as well as OM and zwitterionic DDMG. B, representative section of ¹H^N-¹⁵N correlation spectra of N bound to DDAC and TDAC micelles, resulting in Nα and Nβ states, respectively. C, ¹H^N-¹⁵N correlation spectra of Nβ bound to the micelles indicated. Selected backbone assignments are shown. All spectra were recorded at 35 °C and a ¹H frequency of 700 MHz.

FIGURE 3.

Comparison of structural and dynamic NMR parameter of N bound to different micelles. A, comparison of backbone secondary structure propensities as reflected by secondary ¹³C^α chemical shifts. For ²H/¹³C/¹⁵N-labeled N, random coil conformations are obtained at approximately −0.5 ppm, whereas positive and negative shifts relative to this value denote helical and extended backbone propensities, respectively (45). Shifts for DAC were identical to DDAC and were omitted for the sake of visual clarity. B, comparison of backbone order (pico- to nanosecond time scale dynamics) as reflected by {¹H}-¹⁵N NOE values. Larger values indicate higher order (smaller amplitude H-N amide bond vector dynamics).

FIGURE 4.

Structure of Nβ (DDAC-bound N at pH 5.6). A, superposition of the ensemble of 20 calculated simulated annealing structures. The peptide termini, Met¹–Val⁸ and Lys⁴⁰–Lys⁴², were unstructured. B, schematic representation of the Nβ structure (lowest energy ensemble member). The helical conformation of Ala⁴–Ala⁷ was coincidental to this ensemble member only (see A). C, packing of the β1-β2/α2 transition. The carbon atoms of Ala9 and Ser²⁴ are shown in gray, and the carbon atom of helix α2 residues His²⁵, Glu²⁶, and Ala²⁷ are shown in dark green. Oxygen and nitrogen atoms are shown in red and blue, respectively. D, surface charge distribution color-coded by electrostatic potential. The potential was calculated using APBS (62).

FIGURE 5.

Structural model of Nα (TDAC-bound N at pH 7.4). A and B, model illustrates the helical propensity of α1 and stable helix α2. Destabilization of β1/β2 results in the disruption of any defined β-sheet structure. The relative orientations of β1, β2, and α2 are shown in an arrangement similar to Nβ.

FIGURE 6.

CPT1A structure-based site-directed mutagenesis. Effects of selected point mutations on the sensitivity of full-length rat CPT1A to MCoA inhibition. Maximal inhibition for each construct was determined to be 80% of control activity. The concentrations of MCoA at which half-maximal inhibition (IC₅₀ value) was observed were 56.0 ± 6.8 μm for E3R, 8.1 ± 2.7 μm for G18A, and 1.5 ± 0.3 μm for A9G. The IC₅₀ value for the native rCPT1A was 25.0 ± 4.9 μm. Values are means of 3–5 determinations (± S.E.).

FIGURE 7.

Models of Nβ-CD and Nα-CD associations. A and B, models obtained by docking Nβ to a homology model of the CD using the program ClusPro 2.0 (38). A places the apex of Nβ over the helix formed by Asp⁶⁵⁴–Leu⁶⁶⁷. Arg⁶⁵⁵ of this helix contributes to carnitine binding and is also positioned to interact with MCoA as depicted by dashed lines. B docks Nβ near the CoA-binding site. This model localizes Nβ closer to CD mutations that affect MCoA sensitivity (e.g. Met⁵⁹³ (63)), albeit this may merely reflect their proximity to the CoA-binding site to which MCoA can bind as well. C, model obtained by manually docking Nα to a homology model of the CD. A Glu²⁶–Lys⁵⁶¹ salt bridge was enforced. Nα was oriented to permit Glu³–Lys⁵⁶⁰, Glu³-MCoA(NH₂), and Lys⁴¹–MCoA (3′-phosphate) interactions. The outer mitochondrial membrane is expected at the bottom of the panel, providing a binding surface for Nα and parts of the CD. Nα is shown in red, the N and C domains, making up the catalytic domain (CD), are shown in blue and gray, respectively. Bound MCoA and selected amino acid side chains are illustrated in ball-and-stick representation.