Fig 1.
Domain organization and structure of HCV NS5B and human VAPB.
A. Domain organization of HCV NS5B and differentially dissected proteins used in the present study. B. Crystal structure of NS5B(1–570) (PDB code of 1C2P) with the C-linker over residues 531–570 colored in red. C. Domain organization of human VAPB and differentially dissected proteins used in the present study.
Fig 2.
CD and NMR characterization of HCV NS5B proteins.
A. Far UV CD spectra of C-His-tagged NS5B(1–570) (green), N-His-tagged NS5B(1–570) (red), N-His-tagged NS5B(1–531) and NS5B C-linker (531–570) (blue). B. One-dimensional 1H NMR spectra of NS5B proteins. Purple arrows are used to indicate several up-field NMR peaks characteristic of a well-folded protein. C. 1H-15N HMR HSQC spectrum of the 15N-labeled NS5B C-linker (531–570). The red box is used to indicate HSQC peaks of 5 Gly residues in the C-linker peptide.
Fig 3.
HSQC characterization of the binding between VAPB and NS5B.
A. 1H-15N HSQC spectra of the 15N-labeled soluble VAPB protein (1–195) in the absence (blue) and in the presence of the unlabelled N-His-tagged NS5B(1–570) at a molar ratio of 1:2 (red). In the presence of the unlabelled N-His-tagged NS5B(1–570), most well-dispersed peaks disappeared which were identified to be from the MSP domain while the remaining peaks (red) are from the coiled coil domain, based on our NMR assignments of the human VAPB domains previously published (25–27). B. 1H-15N HSQC spectra of the 15N-labeled VAPB-MSP domain in the absence (blue) and in the presence of the unlabelled C-His-tagged NS5B(1–570) at a molar ratio of 1:4 (red). C. 1H-15N HSQC spectra of the 15N-labeled MSP domain in the absence (blue) and in the presence of the unlabelled N-His-tagged NS5B(1–570) at a molar ratio of 1:2 (red). D. 1H-15N HSQC spectra of the 15N-labeled MSP domain in the absence (blue) and in the presence of the unlabelled N-His-tagged NS5B(1–531) at a molar ratio of 1:4 (red).
Fig 4.
Quantification of the binding between MSP domain and NS5B C-linker.
A. 1H-15N HSQC spectra of the 15N-labeled MSP domain in the absence (blue) and in the presence of the unlabelled NS5B C-linker (531–570) at a molar ratio of 1:1 (green) and 1:3 (red). B. Residue-specific chemical shift difference (CSD) of the MSP domain in the presence of the NS5B C-linker (531–570) at a molar ratio of 1:3. The bars for the residues with the CSD value > 0.03 (average + STD) are colored in red. C. Crystal structure of the MSP domain we previously determined in which cyan is used for indicating Pro and residues with missing HSQC peaks in the free state; green for residues disappeared in the presence of the NS5B C-linker (531–570), and red for the residues with the CSD value > 0.03 at a molar ratio of 1:3. The pink and cyan spheres are used to indicate the sites of the mutations Thr46 (to Ile) and Pro56 (to Ser) respectively, which are associated with familial ALS.
Table 1.
Dissociation constants for the binding of the MSP domain with NS5B C-linker, EphA2 and EphA5 obtained by fitting NMR titration data.
Fig 5.
Characterization of the binding between MSP and Eph LBDs.
A. Far-UV CD spectra of 6 Eph LBDs. 1H-15N HSQC spectra of the 15N-labeled MSP domain in the absence (blue) and in the presence of the unlabelled LBDs of EphA4 (B), EphA6 (C), EphA7 (D) and EphA8 (E) at a molar ratio of 1:1 (green) and 1:4 (red).
Fig 6.
Quantification of the binding between MSP domain and EphA2/EphA5.
1H-15N HSQC spectra of the 15N-labeled MSP domain in the absence (blue) and in the presence of the unlabelled LBDs of EphA2 (A) and EphA5 (B) at a molar ratio of 1:1 (green) and 1:3 (red). C. Residue-specific chemical shift difference (CSD) of the MSP domain in the presence of EphA2 (red) and EphA5 (blue) at a molar ratio of 1:4. Crystal structure of the MSP domain in which cyan is used for indicating Pro and residues with missing HSQC peaks in the free state; green for residues disappeared in the presence of EphA2 (D) or EphA5 (E), and red for the residues with the CSD value > 0.083 for EphA2 and >0.073 for EphA5 at a molar ratio of 1:4. The pink and cyan spheres are used to indicate the sites of the mutations Thr46 (to Ile) and Pro56 (to Ser) respectively, which are associated with familial ALS.