Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling.

TitleIncreasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling.
Publication TypeJournal Article
Year of Publication2015
AuthorsHeselpoth RD, Yin Y, Moult J, Nelson DC
JournalProtein Eng Des Sel
Date Published2015 Apr
KeywordsAlgorithms, Amidohydrolases, Bacteriophages, Computational Biology, Crystallography, X-Ray, Endopeptidases, Enzyme Stability, Histidine, Hydrogen Bonding, Kinetics, Point Mutation, Protein Engineering, Viral Proteins

Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing ΔΔG values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-Å X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by ∼2.2°C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain.

Alternate JournalProtein Eng. Des. Sel.
PubMed ID25740429