FIGURE 3.

Identification of the FnZ peptide binding site and binding orientation on ⁸F1⁹F1 using NMR spectroscopy. A, overlay of the ¹H¹⁵N HSQC spectra of free ⁸F1⁹F1 (black) and of ⁸F1⁹F1 bound to the FnZ (red) and LAGESGET (cyan) peptides. Highlighted residues are mentioned under “Results.” B, chemical shift perturbation map for ¹⁵N-labeled ⁸F1⁹F1 upon binding to the FnZ (top) and LAGESGET (bottom) peptides. Blue and red bars indicate absolute chemical shift differences in backbone amide ¹H and ¹⁵N nuclei, respectively, between free and peptide-bound ⁸F1⁹F1. P, proline residues; ○, unassigned residues; ●, residues previously unassigned but now identified. The approximate location of previously defined secondary structure elements of the F1 modules are shown above. Peaks that disappear upon peptide binding from their chemical shift in free ⁸F1⁹F1 but cannot be assigned in the peptide-bound spectrum are indicated by a gray bar of arbitrary size. C, location of combined chemical shift perturbations (square root of (Δδ_HN² + 0.2Δδ_N²)) on FnZ peptide binding mapped onto the structure of ⁸F1⁹F1 (17) (Protein Data Bank entry 3EJH). Small to large chemical shift changes are indicated by as follows: cyan < blue < yellow < orange < red and Trp⁵⁵³ (gray; prepared using PyMOL) (35).