Figure 2.

Ligand binding by hCNNM4_BAT (100 μM), monitored by 2D ¹⁵N HSQC NMR spectra (zoom on a representative region). (A) Effect of Mg²⁺ addition to a final concentration of 0 mM (black, reference), 10 mM (light blue), 20 mM (blue), and 40 mM (dark blue). Signals shifting or diminishing due to addition of only Mg²⁺ or ADPNP (as in (B)) are marked with letters A or B, respectively, to emphasize differences in the affected signal sets, indicating non-overlapping binding sites. Arrows show the shift direction. (B) Effect of ADPNP addition to a final concentration of 0 mM (black, reference), 570 μM (yellow), 1.14 mM (orange), and 5.7 mM (red). (C) Effect of combined Mg²⁺ and ADPNP addition: Mg²⁺ plus 0 mM ADPNP (black, reference), 0 mM Mg²⁺ plus 5.7 mM ADPNP (orange), 0 mM Mg²⁺ plus 18 mM ADPNP (red), and 10 mM Mg²⁺ plus 5.7 mM ADPNP (blue). Moderate concentrations of ADPNP (5.7 mM) in the presence of Mg²⁺ produce an effect similar to the addition of high concentrations of ADPNP alone (18 mM; a representative signal is marked by an asterisk). The pertaining full spectra are shown in Figure S2A–C. Spurious t₁ noise at ¹H frequencies of ca. 8.2 and 8.4 ppm derive from protons H2 and H8 in the purine ring of ADPNP (see Figure S2D).