Nucleoside diphosphate (NDP) kinase
Dictyostelium discoideum (slime mold) P105G mutant
Subunit interaction is very important for the stability of oligomeric proteins. The energy of interaction between subunits is correlated with the contact area between subunits, calculated from known three-dimensional structures. The nucleoside diphosphate (NDP) kinases represent a good model for studying the effect of quaternary structure on protein stability. NDP kinases are ubiquitous enzymes that catalyze the reversible phosphorylation of nucleoside diphosphates by nucleoside triphosphates. Their main function is thought to be the synthesis of (deoxy) nucleotide triphosphates from ATP. Crystal structures have been revealed for several NDP kinases of various sources in the free form, the phosphorylated intermediate, the complexed with various nucleotides, and natural or engineered mutants. The primary sequences of the NDP kinase are highly conserved throughout evolution but their quaternary structures are not. Typically the prokaryotic ones are tetrameric whereas the eukaryotic ones are hexameric. In both cases, the subunit structure is virtually identical and two subunits assemble into a dimeric structure in a similar way. The contacts between the nucleotides and the protein are remarkably conserved in enzymes from different sources, from bacteria to humans.
In the paper describing this structure, the authors carried out comparative studies on thermal stability and crystal structures of a tetrameric (E. coli) NDP kinase and of a hexameric (Dictyostelium discoideum) NDP kinase, including the P105G mutant, which affects a loop implicated in subunits contacts. Subtle differences are observed between the crystal structures of the wild-type and the P105G mutant enzymes, with the missing proline side chain of the mutant replaced by a water molecule. Calorimetric thermal stability studies show that the minimal change in the structure between the D. discoideum wild-type and P105G mutant enzymes leads to a dramatic change of protein thermal stability. The wild-type enzyme is stabilized by its quaternary structure, such that the thermal transition is observed from a native hexamer to unfolded monomers. In contrast, denaturation of the P105G mutant shows no such dependence, and heating produces first native monomers and then, at higher temperatures, their unfolding. The NDP kinase from E. coli behaves like P105G mutant from D. discoideum.
Protein Data Bank (PDB)
author: Aki Nagata