Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN

Abstract

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (<r.m.s. deviation>bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (<r.m.s. deviation>bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Keywords: apoptosis; cardiolipin; confined space; cytochrome c; lipid-protein interaction; nuclear magnetic resonance (NMR); protein folding; protein hydration; protein stability.

FIGURE 1.

The structure of oxidized horse heart cytochrome c encapsulated in reverse micelles. a, the 32 lowest energy structures from the final round of the simulated annealing protocol in Xplor-NIH (20) are aligned and the secondary structure elements are colored with different shades of blue. The four internal water molecules are displayed as blue dots, and the overlaid heme moieties are highlighted in salmon. b, precision of the NMR-determined model is illustrated by the thickness of the backbone trace as a function of the local r.m.s. deviation, which is also colored from lowest (blue) to largest (cyan) r.m.s. deviation. c, overlays of the backbones of crystal structures of ferricytochrome c determined in low salt (PDB code 1CRC, blue) (39) and high salt (PDB code 1HRC, cyan) (23), a solution structure of cytochrome c (PDB code 1AKK, orange) (40), and that determined by solution NMR methods of the RM-encapsulated protein (PDB code 2N3B, green). All residues were used for the alignment.

FIGURE 2.

Structural and hydration waters of ferricytochrome c. Structural water atoms are colored as semi-transparent cyan spheres. NOEs used to constrain the position of the water are shown with yellow lines. NOE/ROE ratios of amide hydrogen dipolar interactions with water also provides insight into the dynamics of hydration water. NOE/ROE values are plotted as colored spheres ranging from 0 (most dynamic, red) to −0.5 (most rigid, blue). These studies were carried out in cetrimonium bromide/hexanol reverse micelles, which minimizes artifacts arising from hydrogen exchange (18, 38).

FIGURE 3.

Cytochrome c undergoes structural changes upon change in redox state. Violations of the determined magnetic susceptibility tensor at localized sites in the protein likely arise due to differences in structure of the two redox states of cytochrome c. Violations of the back-calculated PCS values are plotted on the structure of encapsulated ferricytochrome c. They range from 0.0009 (thin, red backbone) to 2.075 ppm (thick, blue backbone) with violet indicating intermediate values. Detected redox-dependent changes in structure are localized to the heme-exposed face of cytochrome c, including residues such as Gln-16, Lys-27, and Ile-81 that are in direct contact with BC1, as well as residues 53–56. The RM structure of ferricytochrome c was aligned with its counterpart in complex with the BC1 electron transfer chain binding partner (PDB code 1KYO). Alignment and figure created using PyMol. All residues were used for the alignment.

FIGURE 4.

Identification of cardiolipin interaction sites on ferricytochrome c. The addition of CL to cytochrome c-containing RMs produces specific chemical shift changes in the protein. a, ¹⁵N-HSQC spectra at increasing concentrations of CL. A number of resonances, several of which are boxed separately, are clearly affected by the presence of CL. b, the normalized CSP (55) for each residue with a clearly resolved peak is plotted according to residue number. The normalized CSP = √(Δ¹⁵N/9.8655)² + Δ¹(H)². Negative values indicate that the CSP could not be determined due to various reasons such as cross-peak overlap. c, the chemical shift perturbations rendered onto the surface of the determined structure reveals three interaction sites (no CSP is colored yellow, whereas maximum CSP is blue). Previously suggested interaction sites (A and L) are present in the RM experiments. An additional interaction site is present at a distinct location and is comprised of residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. The side chains of Trp-59 and Asn-52, previously termed the C-site (2), do not display any CSPs even at maximum CL concentration used.