The Ca2+-binding site Ca1–2 is degenerated in the C-terminal domain of the P. polymyxa neutral protease Gentlyase. (a) The double calcium site Ca1–2 is occupied by only one Ca2+ in both structures of P. polymyxa neutral protease. Water molecules coordinating the Ca2+ ion (yellow) are shown as red spheres. (b) Ca1–2 in the prototypic neutral protease thermolysin from B. thermoproteolyticus (PDB entry 3fvp
). (c) Superimposition of Ca1–2 in P. polymyxa neutral protease (yellow) with that of thermolysin from B. thermoproteolyticus (green) shows that the Asp185 coordinating the first Ca2+ in thermolysin is substituted by two water molecules and that Lys172 provides the missing positive charge of the second Ca2+ in P. polymyxa neutral protease. Waters were omitted for clarity.