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Cell-Free Mutant Analysis Combined with Structure Prediction of a Lasso Peptide Biosynthetic Protease B2

  • Almasul Alfi
    Almasul Alfi
    RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
    More by Almasul Alfi
  • Aleksandr Popov
    Aleksandr Popov
    Center for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia
    RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
  • Ashutosh Kumar
    Ashutosh Kumar
    RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
  • Kam Y. J. Zhang
    Kam Y. J. Zhang
    RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
  • Svetlana Dubiley
    Svetlana Dubiley
    Center for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia
    Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
  • Konstantin Severinov
    Konstantin Severinov
    Center for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia
    Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
    Waksman Institute for Microbiology, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, United States
  • , and 
  • Shunsuke Tagami*
    Shunsuke Tagami
    RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
    *Email: [email protected]
Cite this: ACS Synth. Biol. 2022, 11, 6, 2022–2028
Publication Date (Web):June 8, 2022
https://doi.org/10.1021/acssynbio.2c00176
Copyright © 2022 American Chemical Society

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    Abstract

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    Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble, and no experimentally solved structures are available. Here, we performed a rapid semicomprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (FusB2), using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 FusB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, FusB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure–function research of proteins for which no structures are available.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.2c00176.

    • (Figure S1) Expression of FusB2 mutants in the PURE system; (Figure S2) Solubility of FusB2 in the PURE system; (Figure S3) AlphaFold2 predicted structure of FusB2; (Figure S4) Activity test of the catalytic site mutants in the PURE system; (Figure S5) Conformation of the loop between β2 and β3; (Figure S6) AlphaFold2 predicted structure of FusA-Leader·FusB1·FusB2 complex; (Figure S7) Superimposition of the predicted structure of FusA-Leader·FusB1·FusB2 and the crystal structure of FusA-Leader·FusB1; (Figure S8) AlphaFold2 predicted structure of PsmB2; (Table S1) Synthetic genes; (Table S2) DNA oligos for cell-free experiments; (Table S3) PCR schemes for FusA-sfGFP, FusB1, and FusB2 variants; (Table S4) Primers used for in vivo mutation analysis of PsmB2 (PDF)

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    Cited By

    This article is cited by 2 publications.

    1. Ashley M. Kretsch, Mayuresh G. Gadgil, Adam J. DiCaprio, Susanna E. Barrett, Bryce L. Kille, Yuanyuan Si, Lingyang Zhu, Douglas A. Mitchell. Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element. Biochemistry 2023, 62 (4) , 956-967. https://doi.org/10.1021/acs.biochem.2c00700
    2. Kyle E. Shelton, Douglas A. Mitchell. Bioinformatic prediction and experimental validation of RiPP recognition elements. 2023, 191-233. https://doi.org/10.1016/bs.mie.2022.08.050

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