Exploring the Stability and Cardiolipin Affinity of Cytochrome C's Domain Swapped Dimer Conformation

Tuesday, February 14, 2017 coarse-grained molecular dynamics simulations to illuminate the lipid transfer mechanism of CETP in HDL-CETP-LDL ternary complex. The extent of penetration of CETP into HDL and LDL in our ternary complex model corroborated well with the experimental findings. Further, the results demonstrated that the ejection of C-terminal plug-in phospholipid (PLC) and subsequently the diffusion of CETP-bound CEs into LDL droplet through PLC opening. A detailed analysis on interactions of PLC pocket lining residues with the discharged lipids revealed residues with significant role in CE transfer. The detailed mechanism of lipid transfer by CETP as explored in this study might help designing future CETP research and subsequent CETP therapeutics. References: 1. Tall, A.R. Plasma cholesteryl ester transfer protein. J. Lipid Res. 34: 1255– 1274 (1993). 2. Zhang, L. et al., Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein. Nat. Chem. Biol. 8: 342–349 (2012). 1911-Pos Board B231 Role of Bound Phospholipids in Structural Stability and Functionality of Cholesteryl Ester Transfer Protein Prasanna Diddige Revanasiddappa, Revathi Shankar, Sanjib Senapati. Biotechnology, IITM, Chennai, India. Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CEs) and triglycerides (TG) between high density lipoproteins (HDL) and low density lipoproteins (LDL).1 Previous animal model studies have shown that blocking the function of CETP can increase the level of HDL in blood plasma and suppress the risk of cardiovascular disease (CVD). Hence, understanding the mechanism by which CETP transfers the neutral lipids between lipoproteins are of immense importance. In a recent report (Chirasani et al., J. Biol. Chem. 2016), we have shown that the bound CEs intraconvert between bent and linear conformations as a consequence of high degree of conformational flexibility of the protein.2 In this study we explored the importance of bound phospholipids (PLs) in maintaining the structural integrity of CETP and its role in lipid transfer. Results from all atom molecular dynamic simulation show that N-terminal bound PL imparts fluctuations and C-terminal bound PL provides stability to the CETP structure. They are also found to play a crucial role in CE transfer from HDL to LDL. These observations are confirmed by restraining both PLs, which showed conformational rigidity in CE. The detailed insights obtained here on the role of PLs in CETP could help in devising methods to prevent the CETPs function in countering atherosclerosis. References 1. Mistry, Q. X., et al., Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nat. Struct. Mol. Biol. 14, 106-13 (2007) 2. Chirasani, V. R., et al., Structural Plasticity of Cholesteryl Ester Transfer Protein Assists the Lipid Transfer Activity. J. Biol. Chem. 291, 19462-19473 (2016) 1912-Pos Board B232 Oxidation State and PH Dependence of Cytochrome C Binding to Cardiolipin-Containing Liposomes Bridget Milorey, Reinhard Schweitzer-Stenner. Drexel University, Philadelphia, PA, USA. A combination of UV/visible absorption, circular dichroism and fluorescence spectroscopies was used to probe the binding of cytochrome c to cardiolipin (CL) containing liposomes and concomitant conformational changes as a function of cardiolipin concentration at different pH values. Our group has recently developed a model that describes oxidized cytochrome c binding to CL-containing liposomes as a two-step process where native-like liposome bound conformers convert into more unfolded conformations. We have provided evidence that the M80 ligand in the more unfolded state has been replaced by either a lysine or histidine as the axial ligand. A slightly modified binding model was used to describe the binding of reduced cytochrome c, to account for the redox properties of the heme in the absence and presence of oxygen. In our current study, we explored how lowering the pH to values between 6.0 and 7.0 affects binding and conformation of cytochrome c in both oxidation states. It has recently been proposed that such a protocol would facilitate the so-called L-site binding, which involves the electrostatic interaction of amino acid residues Lys22, 25, 27 and His26 and 33 with the acidic phospholipids on the liposome surface. Our results for binding at pH 6.5 indicate a conversion from a low spin to a hexacoordinated high-spin state at moderate to high cardiolipin concentrations, which leads to a significant blue shift of the Soret band. This very much resembles the behavior of denatured cytochrome c where lowering the pH leads to the protonation of a distal histidine ligand and its replacement by a water ligand. Thus, our observations strongly suggest that histidine rather than lysine is the sixth ligand in the misligated state of cytochrome c on CL-containing liposomes.

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1913-Pos Board B233 Alanine Charge Screening Demonstrates Cytochrome C Unfolding on Cardiolipin Membrane Surfaces Margaret M. Elmer-Dixon, Bruce E. Bowler. University of Montana, Missoula, MT, USA. Cytochrome c (Cytc) has been shown to play an important role in signaling during apoptosis. In the initial stages, positively charged regions of Cytc interact electrostatically with negatively charged, cardiolipin (CL) on the inner mitochondrial membrane. In the presence of reactive oxygen species, Cytc demonstrates increased peroxidase activity and a propensity to oxidize CL. Cytc dissociate from the oxidized CL freeing it to form the apoptosome in the cytoplasm and initiate apoptosis. The exact mechanism for this initial electrostatic interaction and the role of structure and conformation of Cytc have been extensively debated. Using heme Soret band circular dichroism (CD), fluorescence (Trp59) and UV-visible spectroscopies, in coordination with alanine charge screening, we investigate the initial electrostatic cytochrome c/cardiolipin interaction. To date, several cardiolipin interaction sites have been proposed on cytochrome c. At pH 8, we are able to isolate and study the cytochrome c/cardiolipin interaction at anionic binding site A with 100% cardiolipin vesicles. Using alanine charge screening (Lys/Ala mutations), we investigated the role of lysines 72, 73, 86 and 87 in site A during electrostatic cardiolipin binding. Optimized titration techniques were utilized that generate reproducible quantitative data that can then be fit to extract the binding contribution to cardiolipin for each amino acid investigated. Our data indicate that an initial binding occurs at lipid to protein, L/P, ratios below 5. However, neither Trp59 fluorescence nor Soret band CD is strongly sensitive to this early binding event making quantitative evaluation of this interaction difficult. At L/P ratios >20 a binding event consistent with a conformational rearrangement on the surface of the CL vesicle occurs. Our data indicate that one lysine from either side of Site A (Lys72/73 versus Lys86/87) is important for both the cooperativity of and L/P ratio needed for this conformational rearrangement on the liposome surface. 1914-Pos Board B234 Exploring the Stability and Cardiolipin Affinity of Cytochrome C’s Domain Swapped Dimer Conformation Harmen B. Steele1,2, Levi J. McClelland1,2, J.B. Alexander Ross1,2, Bruce E. Bowler1,2. 1 Biochemistry and Biophysics, University of Montana, Missoula, MT, USA, 2 Chemistry & Biochemistry, University of Montana, Missoula, MT, USA. Cytochrome c’s (cytc) oxidation of the lipid cardiolipin (CL) is a proposed switch involved in initiation of the intrinsic pathway of apoptosis. Recently, there has been an increased number of reports in the literature of cytc domain-swapped dimers (DSD). The Hirota lab has reported that the horse DSD has increased peroxidase activity compared to the monomeric form. The Bowler lab has solved DSD crystal structures with various detergents binding in the hydrophobic tube of the proposed ‘‘C’’ cytc-CL binding site. We postulate that the DSD conformation of cytc is an evolutionary switch allowing for tighter regulation of the intrinsic pathway of apoptosis. In order to examine the stability of the DSD, samples were incubated temporally at fixed temperatures and then analyzed using size-exclusion chromatography. The data, presented here, indicate that the human DSD is more kinetically stable than horse or yeast DSD, suggesting evolution of a more kinetically stable conformation in primates. Additionally, fluorescence correlation spectroscopy (FCS) data, also presented, show the binding of Zn-cytc to CL nanodiscs (ND). ND are discoidal lipid bilayers maintained by two alpha helical belt proteins and provide a nice model system to study protein-lipid interactions. Initial FCS results indicate that the Kd of cytc for CL NDs is in the micromolar range. This poster will compare the relative CL binding affinities of monomer and DSD cytc. 1915-Pos Board B235 Selective Probing of Non-Native Cardiolipin-Bound Conformations of Ferricytochrome C via Ferrocyanide-Mediated Photoreduction Dmitry Malyshka, Reinhard Schweitzer-Stenner. Chemistry, Drexel University, Philadelphia, PA, USA. Upon binding to cardiolipin (CL), ferricytochrome c replaces its native Met80 ligand and ultimately gains peroxidase activity, a vital step towards its dissociation from the inner mitochondrial membrane and its subsequent initiation of apoptosis. Multiple binding studies involving ferricytochrome c and CL-containing liposomes have resulted in a variety of partially conflicting binding models, but little quantitative insight into the co-existence of native and partially unfolded conformations of ferricytochrome c has been obtained thus far. While multiple groups have put forth binding models to explain this