Mandal Abhishek, Hoop Cody L, DeLucia Maria, Kodali Ravindra, Kagan Valerian E, Ahn Jinwoo, van der Wel Patrick C A
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Departments of Environmental and Occupational Health, Chemistry, Pharmacology, and Chemical Biology and Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, Pennsylvania.
Biophys J. 2015 Nov 3;109(9):1873-84. doi: 10.1016/j.bpj.2015.09.016.
The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.
内源性凋亡的细胞过程依赖于线粒体脂质的过氧化作用作为关键分子信号。脂质过氧化与线粒体活性氧的增加相关，但线粒体细胞色素c（cyt-c）也发挥着必要作用。在凋亡的线粒体中，cyt-c获得了一种新功能，即作为脂质过氧化物酶催化活性氧介导的线粒体脂质心磷脂（CL）的化学修饰。这种过氧化物酶活性是由蛋白质的构象变化引起的，该变化源于cyt-c与CL之间的相互作用。构象变化的本质及其如何导致这种功能获得仍不确定。通过功能、结构和生物物理实验的结合，我们研究了膜结合状态下cyt-c的结构和过氧化物酶活性。我们用含CL的脂质囊泡重构了cyt-c，并确定了膜结合导致的过氧化物酶活性增加。我们将这些CL诱导的促凋亡活性测定与通过固态核磁共振和光谱学对膜结合蛋白的结构和动力学研究相结合。使用均匀（13）C、（15）N标记蛋白的多维魔角旋转（MAS）固态核磁共振来检测与含CL脂质双层结合的氧化型和还原型马心cyt-c的位点特异性构象变化。MAS核磁共振和傅里叶变换红外测量结果表明，外周膜结合的cyt-c经历了显著的动力学变化，但仍保留了大部分或全部二级结构。此外，在二维和三维MAS核磁共振谱中，与CL结合的cyt-c显示出光谱分辨率，因此具有结构均匀性，这与广泛的膜诱导展开不一致。发现cyt-c主要与膜界面相互作用，而不会显著破坏脂质双层。因此，膜结合导致cyt-c获得增加的过氧化物酶活性，这代表了其关键的促凋亡功能，但我们没有观察到大规模展开或渗透到膜核心的证据。
