Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Abstract

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

Keywords: lipid peroxidation; lipidomics; mitochondrial apoptosis; peripheral membrane proteins; solid-state NMR spectroscopy.

Figure 1.. Cytochrome c and its anionic lipid partners.

(a) Cartoon representation of cyt c secondary structure, showing the five foldons [34]: the blue foldon (N and C- terminal helices, residues 1–14 and 88–104), the green foldon (helix 61–69 and Ω loop 20–35), the gray foldon (Ω loop 40–57), the red foldon (Ω loop 71–85) and the yellow unit (residues 37–40 and 57–60). (b) 3D structure of horse heart cyt c [35] (PDB ID: 1hrc) colored and labeled according to the different foldons [10]. The dotted red circle marks the “site A” lipid binding site. (c) Chemical structure and enzymatic interconversion of phosphatidylglyerol (PG) and CL, catalyzed by CL synthase. (d) Comparison of headgroup net charge, lipid area, liquid to gel phase transition temperature (T_m) and the acyl chain information of TOCL and DOPG [–38].

Figure 2.. Binding and peroxidase activation by PG and CL containing lipid vesicles.

(a) Fraction of membrane-bound cyt c as a function of outer layer anionic lipid/cyt c ratio (“effective” anionic lipid/cyt c ratio). The molar fraction of PG or CL in the DOPG/DOPC and TOCL/DOPC vesicles is indicated. Saturation of cyt c binding to DOPG requires approximately twice the lipids as for TOCL. (b) Comparison of fractional oxygenation of polyunsaturated PG and CL by cyt c/H₂O₂ quantified by MS lipidomics. The molar ratio of PG to cyt c was kept at 25:1 and CL to cyt c at 12.5:1 so that the total phosphate to cyt c ratio was the same. The effective anionic lipid/cyt c ratio was 12.5 for PG and 6.3 for TOCL. The concentration of cyt c was kept at 1 μM, and H₂O₂ at 50 μM. Data are presented with mean value and SD.

Figure 3.. Structural similarities and dynamic perturbations in PG- and CL-bound cyt c.

(a) Region from a 2D ¹³C-¹³C CP-DARR ssNMR spectrum showing correlations of backbone Ca with sidechain carbons, and (b) 2D ¹⁵N-¹³C ssNMR spectrum, for GIL-labeled cyt c bound to DOPC LUVs containing 25% TOCL (yellow) or 50% DOPG (black). GIL-labeled cyt c is selectively labeled with ¹³C, ¹⁵N- Gly, Ile, and Leu. The total L/P molar ratio is 50 for both, with effective molar PG:cyt c and CL:cyt c ratios of 12.5 and 6.25, respectively. Residues showing prominent peak intensity perturbations are labeled in green. (c) Chemical shift perturbations for GIL-labeled cyt c between the DOPG and TOCL-bound protein, sensing differences between the two lipid complexes. G56 of loop C shows significant perturbation. Helices and foldon units are indicated atop the chart, with colors as in Fig. 1. (d) Horse heart cyt c structure [35], with perturbation sites labeled in green, while orange highlights indicate NMR-observed residues without significant perturbations. The perturbed residues belong to loops B, C and D.

Figure 4.. Probing the tertiary fold of cyt c bound to CL and PG LUVs.

(a) Extracted regions in 2D ¹³C-¹³C PDSD ssNMR spectra (mixing time 800 ms) showing interhelical contacts between G6 at the N terminus and L94 at the C terminus. The spectrum of cyt c bound to (1:3) TOCL/DOPC liposomes at a LP of 50 is shown in orange and that bound with (1:1) DOPG/DOPC at the same L/P ratio shown in black. Bottom: 1D slices at the position of G6 Cα show inter-residue correlations with L94 and I9. Spectra were acquired at 265 K, MAS of 10 kHz, and 17.6 T. (b) The N- and C- terminus contacts between G6 and L94 detected are mapped on the cyt c structure in green dashed lines (PDB ID: 1hrc; [35]). (c) CL-induced dynamic changes of cyt c detected by fluorescence spectroscopy. The dynamic changes induced by membranes increase in the order of 50% DOPG, 25% TOCL, and 50% TOCL, and a higher L/P ratio induces more dynamic changes than the lower. Three replicates are presented in the plot. Error bars are within the data point symbols.

Figure 5.. Increased local dynamics for membrane-bound cyt c at higher L/P ratio.

2D ¹³C-¹³C CP-DARR ssNMR spectrum of GIL-labeled cyt c bound to (1:1) TOCL/DOPC vesicles: (a) at an effective CL/cyt c ratio (R) of 25 measured at 256 K; (b) measured at 240K; (c) and at a CL/cyt c ratio of 6.3 at 256 K. The greater excess of CL leads to loss of signals in part of the protein, compared to near-saturating conditions. Suppressing the underlying dynamics at lower temperature (b) leads to recovery of the missing peaks. (d) Chemical denaturation with 6 M urea of membrane-bound cyt c causes the observed signals to feature a narrow chemical shift dispersion and much broader linewidths, consistent with an unfolded state. Urea sample studied at 240 K using (1:1) TOCL/DOPC liposomes at a L/P ratio of 25. All spectra were obtained at 750 MHz (¹H) and 10 kHz MAS.

Figure 6.. CL binding impacting cyt c foldon dynamics.

(a) 2D ¹³C-¹³C ssNMR spectrum of GIL-labeled cyt c bound to (1:1) TOCL:DOPC liposomes at a L/P ratio of 100 (maroon) overlaid with that of cyt c bound to (1:3) TOCL:DOPC liposomes at L/P= 50 LP (black). The effective CL/P ratio R is 25 and 6.3, respectively. Both spectra were acquired at 256 K, 10 kHz, and a magnetic field of 15.6 T. Select residues lack signals in the high CL/P spectrum; the remaining residues are labeled. Those showing intensity and chemical shift perturbations are labeled in red and yellow, indicating the red and yellow foldons. (b) The blue foldon (N-, C-helices) and 60’s helix of the green foldon experience only slow motion at a L/P ratio of 100 (top). Heme-binding Ω loop (red) and 57–60 loop (yellow) exhibit intermediate dynamics (middle). Green foldon (20–35 Ω loop) shows faster dynamics rendering their NMR signals invisible (bottom). (c) PDSD transfer build-up curves for one-bond cross-peaks (C_α-C’) of α-helix and loop residues, for cyt c bound to (1:3) TOCL:DOPC liposomes at a 50 L/P, acquired at 256 K, 10 kHz, and a magnetic field of 15.6 T. Residues I9, L64, and I95 of helical regions are plotted in purple, and loop residues L32, I57, and G84 in black. The loop residues show increased mobility compared to those in the helices, based on differences in these curves (see text).

Figure 7.. Hierarchical dynamics of folding units of cyt c upon binding to CL-containing membranes.

(a) Overview of hierarchical dynamics detected by ssNMR. (b) Molecular structure of cyt c highlighting key residues and segments. The dashed line indicates the hypothesized opening of the CL-bound protein fold, such that it would preserve the stable blue foldons with associated heme (top/left), while increasing the spacing between heme and residues Met80, Phe82 and Trp59 (right/bottom), as detected spectroscopically. The range of motion depends on the experimental conditions (e.g. lipid composition).