Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase.

Sanjeev Kumar,Kallol Gupta,Aparna Dongre,Hemalatha Balaram,Padmanabhan Balaram,Sunita Prakash

doi:10.1038/ncomms12798

Abstract

Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii. By comparing the biophysical properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability.

Highlights

Protein ageing is often mediated by the formation of succinimide intermediates
Loss of NH3 from N109 leading to succinimide formation would result in lowering of mass by 17 Da, while hydrolysis of the succinimide to aspartic acid (Asp) or isoaspartic acid would lead to an increase in mass by 1 Da over that expected from the sequence of this peptide
Both peptides (1,352.7 and 1,370.7 Da) were subjected to fragmentation by matrix-assisted laser desorption ionization (MALDI), collision-induced dissociation (CID) and electron transfer dissociation (ETD)-MS/MS to validate the site of succinimide formation (Fig. 1c,d and Supplementary Fig. 2b–e)

Summary

Introduction

Protein ageing is often mediated by the formation of succinimide intermediates. These shortlived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into b-aspartyl or D-aspartyl residues. Studies on the mechanism of deamidation/dehydration of Asn/Asp residues in proteins indicate that succinimide is metastable and the only functional role attributed to this intermediate is in protein splicing[18,19]. This apart, no other evidence for a biochemical or structural role has been associated with the succinimidyl intermediate. We report on the role played by the post-translational modification of a specific asparaginyl residue, N109 to succinimide in Methanocaldococcus jannaschii glutaminase (MjGATase) in imparting structural stability to the enzyme. We demonstrate that the succinimide is indispensable for the structural stability and the enzymatic activity of MjGATase at elevated temperatures

Methods

Results

Conclusion