Structural basis for the hyperthermostability of an archaeal glutaminase induced by post-translational succinimide formation

Stability of proteins from hyperthermophiles enabled by reduction of conformational flexibility is realized through various mechanisms. Presence of a stable, hydrolysis-resistant succinimide arising from cyclization of the side chains of aspartyl/asparaginyl residues with backbone amide -NH of the succeeding residue would restrain the torsion angle {Psi}. Here, we describe the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and address the mechanism of a succinimide-induced increased thermostability using molecular dynamics simulations. This study reveals the interplay of negatively charged electrostatic shield and n[->]{pi}* interactions in preventing succinimide hydrolysis. The stable succinimidyl residue induces formation of a conformational-lock, reducing protein flexibility. Protein destabilization upon replacement with the {Phi}-restricted prolyl residue highlights the specificity of the conformationally restrained succinimidyl residue in imparting hyperthermostability. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in a group of archaeal GATases suggests an adaptation of this otherwise detrimental post-translational modification as an inducer of thermostability.