Fig. 2

Determination of the histidine residues implicated in Zn²⁺ coordination. a ¹H-¹⁵N HSQC spectrum of 250 μM ¹⁵N- and ¹³C-labeled Pgm(692–768)* at 800 MHz, in the presence of 5 mM Hepes pH 6.8, 25 mM NaCl at 20 °C. Pgm(692–768)* corresponds to residues 692–768 plus the first seven linker residues (in blue, see also panel e) left after PreScission cleavage of the GST-Pgm(692–768) fusion. b Schematic representation of the two tautomeric states of histidine able to complex zinc ions. The Nδ1 coordination form is associated to the H-Nε2 tautomeric form and the Nε2 coordination form is associated to the H-Nδ1 tautomeric form. Zinc ions are represented as red spheres. c ¹H-¹³C HSQC (20 °C, pH 7.5) used to compare the Cδ2, Hδ2 and Cε1, Hε1 chemical shifts of His701, His738 and His749. d ¹H-¹⁵N HSQC (20 °C, pH 7.5) used to compare the Nδ1 and Nε2 chemical shifts of His701, His738 and His749 e Sequence of the Pgm(692–768)* peptide used for NMR experiments. The Zn²⁺ ligands are highlighted in red. TALOS+ secondary structure predictions based on the experimental chemical shift information and those found in the 3D NMR structures of Pgm(692–768) are shown, with β-strands in green (703–705, 710–712, 718–719, 722–724) and α-helices in orange (725–730 and 745–751). The additional mini-helix observed in the final structures is highlighted in blue. The cross-brace arrangement of the two zinc-binding motifs is indicated by blue and pink arrows, each corresponding to one zinc-binding motif