NMR Analysis Suggests Synergy between the RRM2 and the Carboxy-Terminal Segment of Human La Protein in the Recognition and Interaction with HCV IRES

Figures

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  Figure 1. (A) 1H-15N HSQC spectrum and assignment of human La RRM2-Cter; (B) chemical shift mapping of the unassigned residues’ NHs (yellow) of the CS Rosetta model of RRM2-Cter.

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  Figure 2. (A) heteroNOE values of RRM2-Cter (La 224–408); (B) R2/R1 and R1 × R2 values of RRM2-Cter (La 224–408). Residues with extremely high values of R1 × R2 and/or R2/R1 are noted.

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  Figure 3. (A) Ribbon representation of the affected amino acids mapped into the hRRM2-SBM structure; (B) chemical shift perturbation of 15N-hRRM2-SBM upon the addition of the unlabeled ligand in threshold value 0.076. Red bars indicate CSPs above threshold, while blue bars illustrate CSPs beyond threshold line.

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  Figure 4. (A) Ribbon representation of the affected amino acids mapped into the hRRM2-Cter structure; (B) chemical shift perturbation of 15N-hRRM2-Cter upon the addition of the unlabeled ligand in threshold value 0.064. Red bars indicate CSPs above threshold, while blue bars illustrate CSPs beyond threshold line.

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  Figure 5. (A) Ribbon representation of the affected amino acids mapped into hRRM1 (i) (PDB ID: 1S79) and the 3D structure of hRRM2-Cter (ii) predicted by CS Rosetta; (B) chemical shift perturbation of 15N-hRRM1-RRM2-SBM upon the addition of the unlabeled ligand in threshold value 0.04. Red bars indicate CSPs above threshold, while blue bars illustrate CSPs beyond threshold line.

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  Figure 6. (A) Ribbon representation of the affected amino acids mapped (in orange) into the hRRM2 structure (PDB ID:1OWX); (B) chemical shift perturbation of 15N-hRRM2 upon the addition of the unlabeled ligand in threshold value 0.018. Red bars indicate CSPs above threshold, while blue bars illustrate CSPs beyond threshold line.

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  Figure 7. ITC analysis of the interactions between the human La polypeptides (RRM2-SBM, RRM2-Cter, RRM1-RRM2-SBM and RRM1-RRM2-Cter) and IRES-HCV domain IV. The upper graph in each plot displays the raw data, and the lower graph displays the intergraded heat per injection (circles) and the best fit function (solid line). KD values are presented for each interaction.