Figure 6. Solution NMR analysis reveals manganese‐binding region in the N‐terminal domain of YqgP.

• A
  Solution NMR structure of cytosolic N‐terminal domain of YqgP (YqgP_NTD, amino acids 1–177), showing ensemble of 30 structures with lowest energy, and the structure with minimum energy displayed as cartoon.
• B
  Electrostatic surface of YqgP_NTD structure reveals highly negatively charged areas. Left view is oriented as the left view in panel (A).
• C
  Chemical shift perturbation values and relative peak intensities for backbone resonances (¹⁵N and ¹H) of YqgP_NTD calculated for complexes of 400 μM YqgP_NTD with 10 μM (light grey, light orange) or 40 μM MnCl₂ (dark grey, dark orange bars). Only residues 20–100 of YqgP are displayed for simplicity. Residues identified as potential manganese‐binding region based on their chemical shifts and intensity changes upon NMR titrations are depicted in orange. The X sign marks residues whose chemical shifts were not calculated due to the lack of resonance signal.
• D
  Details of 2D ¹⁵N/¹H HSQC spectra of free (green) and Mn‐titrated YqgP_NTD (10 μM MnCl₂ in red and 40 μM MnCl₂ in blue) for the residues marked in orange in panel (C). As a reference, residues marked in black do not display spectral changes upon titration, e.g. peak intensity does not change for S88N‐H upon Mn titration (overlay of green, blue and red contours), but peak intensity is severely depleted for D29N‐H upon Mn titration (absence of red contours). Full titration data are available in Dataset EV3.
• E, F
  Detection (E) and quantification (F) of steady‐state conversions of endogenous MgtE by YqgP variants bearing single‐point mutations in putative Mn‐binding region (BS184; 187; 196–203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl₂. Black arrow marks full‐length MgtE, and red arrow marks its N‐terminal cleavage product by YqgP.
• G
  Overview of residues affected by Mn²⁺ binding to YqgP_NTD mapped onto its solution NMR structure. The view is oriented as the left view in panels (A and B).