Surface salt bridges contribute to the extreme thermal stability of an FN3‐like domain from a thermophilic bacterium

Abstract

This study uses differential scanning calorimetry, X‐ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3‐like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three‐stranded beta sheet packing against a four‐stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm −19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

Computation‐Based Design of Salt Bridges in PETase for Enhanced Thermostability and Performance for PET Degradation

“…Unexpectedly, the distances between the O − atom of D186 and the N + atom of R168 were less than 4.0 Å in both variants, which indicated that the salt bridge interaction between R168 and D186 was also formed in the variants. The dynamics of the salt bridge interaction in the variants were then analyzed based on the distance between the CZ atom of Arg and the CG atom of Asp/the CD atom of Glu (Figures 7e and 7 f) [16a] . The salt bridge interaction between R168 and D186 in both variants could be formed after 10 ns (CZ‐CG distance <5.0 Å), [16a] and a more stable salt bridge was formed with a shorter distance (4.0 Å) in Is PETase I168R/S188E .…”

“…The dynamics of the salt bridge interaction in the variants were then analyzed based on the distance between the CZ atom of Arg and the CG atom of Asp/the CD atom of Glu (Figures 7e and 7 f) [16a] . The salt bridge interaction between R168 and D186 in both variants could be formed after 10 ns (CZ‐CG distance <5.0 Å), [16a] and a more stable salt bridge was formed with a shorter distance (4.0 Å) in Is PETase I168R/S188E . The mean distance between the CZ atom of R168 and the CG atom of D188 in Is PETase I168R/S188D was 5.5 Å in the last 2.5 ns, while the mean distance between the CZ atom of R168 and the CD atom of E188 in Is PETase I168R/S188E was 4.5 Å in the last 5.0 ns.…”

Computational thermostability engineering of a nitrile hydratase using synergetic energy and correlated configuration for redesigning enzymes (SECURE) strategy

“…Electrostatic interactions comprising hydrogen bonds 52 and salt bridges 53 were verified to significantly contribute to the protein stability. The replacement of A71D, A74D, and A78R located in the α-helix caused changes in the charge distribution on the α-subunit surface, accompanied with the formation of a new salt bridge, i.e.…”

An Unusual Effect of Hofmeister Series Salts on the Stability of Toxoplasma gondii Ferredoxin NADP+ Reductase

“…As we know the proteins from thermophilic organisms have a higher number of surface salt bridges, which helps to stabilize them at higher temperatures. [29,30]. It's possible that the relocalization of ionizable amino acids from the inside of proteins to the surface and their involvement in the development of surface salt bridges explains why acidic pH improves TgFNR's thermal stability.…”