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

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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/432556v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f3f1c7org.highwire.dtl.DTLVardef@1866956org.highwire.dtl.DTLVardef@16dadaforg.highwire.dtl.DTLVardef@1a5c519_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIA mitochondrial protein-lipid complex regulates lipid peroxidation in apoptosis. C_LIO_LIPeroxidase-active lipid-cytochrome c complexes are reconstituted in vitro. C_LIO_LIPhosphatidylglycerol lipids are less effective activators than cardiolipin. C_LIO_LIActivity correlates to localized dynamics, distinct from chemical denaturation. C_LIO_LIA dynamic interplay of cytochrome c foldons and anionic lipids regulate activity. C_LI