Post-translational modifications usually occur under enzymatic control as a mechanism for regulation of protein activity or localization, but can also occur spontaneously during protein aging and degradation. In contrast, there are few examples of spontaneous post-translational modifications with a significant role in the stability or biochemical function of a bacterial protein. Here we show that a spontaneous post-translational modification is structurally encoded and functionally important in a bacterial solute-binding protein. We show that the solute-binding protein SAR11_0655 from the abundant SAR11 marine bacterium Pelagibacter ubiqueversans contains an unusually stable succinimide residue important for its function in high-affinity binding of the amino acid derivative 5-oxoproline. Spontaneous cyclization of Asn269 to form a succinimide residue was inferred from high-resolution X-ray crystallography and confirmed by liquid chromatography-tandem mass spectrometry. Loss of the succinimide modification via the substitution N269A resulted in a >20 {degrees}C decrease in thermostability and a 15 to 50-fold decrease in binding affinity for 5-oxoproline. Analysis of sequence conservation indicated that both Asn269 and Asp269 are common among SAR11_0655 homologs, and the N269D variant of SAR11_0655 also showed partial formation of succinimide. These results reveal a previously unrecognized mechanism by which proteins exploit spontaneous post-translational chemistry as a design feature to enhance stability and function, and provides a potential basis for identification and design of structurally encoded succinimide residues for engineering of protein thermostability.
Clifton, B. E., Akdavletov, B., Jain, P., Laurino, P.
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