engleski

Importance of the disulfide link in the stability of cytosolic protein

We have recently solved the crystal structure of Arabidopsis thaliana cytosolic seryl-tRNA synthetase (SerRS), that is involved in the essential cellular process of translation (protein biosynthesis). Arabidopsis SerRS displays structural features typical of canonical SerRSs, except for a unique intrasubunit disulfide bridge. Interestingly, cysteines (at positions 213 and 244) involved in disulfide link are conserved in all SerRSs from green plants, indicating their plant-specific functional importance. Disulfide link may play a role in the structural stabilization of the SerRS catalytic core or in the allosteric regulation of SerRS activity. We have prepared mutant SerRS proteins with mutation of cysteine into serine: single mutant C213S, single mutant C244S and double mutant C213S_C244S. Using nanoDSF and DSC, thermal stability of wild type and mutant proteins was examined in the presence of various reducing and oxidizing reagents (β-mercaptoethanol, DTT, H2O2, diamide), as well as SerRS substrates (serine, ATP, tRNASer). As expected, C213S mutant showed lower stability compared to the wild type which can be attributed to the fact that it cannot form disulfide link. Unexpectedly, C244S mutant showed higher stability than wild type, although it cannot form disulfide link either. We have solved the crystal structure of the C244S mutant and it appears that serine at position 244 can interact with nearby residues, possibly stabilizing the structure. Interestingly, double mutant had the same stability as the wild type. It appears that mutation C213S that confers lower stability and C244S that confers higher stability compensate each other in the double mutant. The results imply that cysteines involved in the disulfide link are important but not essential for Arabidopsis SerRS stability. Future studies are aimed at determination of enzyme activity of the wild type and mutant proteins in order to decipher whether the disulfide link shows an impact on Arabidopsis SerRS activity. Considering that disulfide bonds in cytosolic proteins are usually linked to cellular response mechanisms to oxidative stress, the disulfide link in plant SerRSs may be involved in a regulation of translation under conditions of oxidative stress.

disulfide link ; thermal stability ; aminoacyl-tRNA synthetase

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