Deprotonated imidodiphosphate in AMPPNP-containing protein structures

Abstract

Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5'-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine.

Figure 1

The molecule of AMPPNP and its environment in the following proteins: (a) thymidylate kinase (PDB entry 1nn5), (b) tRNA-dependent kinase (3a4l), (c) myosin domain (3myk), (d) topoisomerase (1mx0), (e) adenylate kinase (1ank), (f) EphA kinase (3fxx). The H atoms are not present in the PDB files and are not shown here, but their locations can be inferred from the type of atoms engaged in hydrogen bonds. The figures were created with PyMOL (DeLano, 2002 ▶).

Figure 2

The anion of AMPPNP as it exists in aqueous solution and its complex formed with Mg²⁺ after deprotonation of the β,γ-bridging imide.