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

Protein Engineering, Design & Selection : PEDS
Ryan D HeselpothDaniel C Nelson

Abstract

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. ...Continue Reading

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Citations

Oct 16, 2015·PloS One·Blake T RileySheena McGowan
May 31, 2019·Antibiotics·Xiaoran Shang, Daniel C Nelson
Jul 16, 2017·The Journal of Biological Chemistry·Aron BroomElizabeth M Meiering
Sep 3, 2020·International Journal of Pharmaceutics·Wing Ching Bianca LaiSharon Shui Yee Leung
Feb 3, 2021·Current Opinion in Biotechnology·Sara B LindenDaniel C Nelson
Oct 29, 2019·Journal of Chemical Information and Modeling·Xingrong FangLi Xu

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