Fig. 4: Structural analysis of the interaction between APO and Nt-hMLKL.
A NMR analysis of Ox-APO binding to Nt-hMLKL. APO was pre-incubated for 0, 2, 4, 8, 12, and 24 h at 25 °C for auto-oxidation, and the NMR 1H-15N HSQC spectrum of Nt-hMLKL in the presence of APO (red) is superimposed on that of unbound Nt-hMLKL (blue). The labeled amino acid residues indicate an increase in CSPs due to their interaction with Ox-APO. B CSPs (histogram, left y-axis) and intensity ratio values (dot, right y-axis) of backbone amides of Nt-hMLKL in response to Ox-APO are plotted. α1 to α6 indicate the helices of Nt-hMLKL. I/I0 the intensity ratio, I the intensity of Nt-hMLKL with APO, I0 the intensity of unliganded Nt-hMLKL; green bar, amino acid residues overlapped by CSP peaks that are completely or barely visible. C CSP mapping onto the 3D conformer (PDB ID code 6ZPR). The UCSF Chimera program displays conformers in 3D as ribbon or sphere surfaces and colors amino acid residues from purple (highest) to white (lowest) based on the height of the CSP peaks. Residues not visible by NMR are colored black. D Comparison of the APO binding site of Nt-hMLKL (green; PDB ID code 6ZPR). Ox-APO (yellow) is shown as sticks. Key interacting residues are shown as sticks (covalent bond between Cys86 and Ox-APO; π–π stacking between Phe148 and Ox-APO core). E, F SPR analysis. Nt-hMLKLC86A and Nt-hMLKLF148A (E), and Nt-hMLKLC86A/L89A/D94A and Nt-hMLKLR145A/R146A/F148A/M150A (F) were immobilized on CM5 chips, and Ox-APO at concentrations of 0, 12.5, 25, 50, 100, and 200 μg/mL was flowed through to observe binding.
