Challenges in the Use of Sortase and Other Peptide Ligases for Site-specific Protein Modification

Overview

Journal Chem Soc Rev

Specialty Chemistry

Date 2022 May 5

PMID 35510539

Authors

Holly E Morgan

W Bruce Turnbull

Michael E Webb

Affiliations

Soon will be listed here.

Abstract

Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.

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References

Schmohl L, Bierlmeier J, von Kugelgen N, Kurz L, Reis P, Barthels F . Identification of sortase substrates by specificity profiling. Bioorg Med Chem. 2017; 25(18):5002-5007. DOI: 10.1016/j.bmc.2017.06.033. View

Takeuchi Y, Noguchi S, SATOW Y, Kojima S, Kumagai I, Miura K . Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor). Protein Eng. 1991; 4(5):501-8. DOI: 10.1093/protein/4.5.501. View

Guimaraes C, Witte M, Theile C, Bozkurt G, Kundrat L, Blom A . Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc. 2013; 8(9):1787-99. PMC: 3943461. DOI: 10.1038/nprot.2013.101. View

Morrison P, Balmforth M, Ness S, Williamson D, Rugen M, Turnbull W . Confirmation of a Protein-Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase-Mediated Labelling. Chembiochem. 2016; 17(8):753-8. PMC: 5963676. DOI: 10.1002/cbic.201500547. View

Wang H, Altun B, Nwe K, Tsourkas A . Proximity-Based Sortase-Mediated Ligation. Angew Chem Int Ed Engl. 2017; 56(19):5349-5352. PMC: 5537000. DOI: 10.1002/anie.201701419. View

Race P, Bentley M, Melvin J, Crow A, Hughes R, Smith W . Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism. J Biol Chem. 2009; 284(11):6924-33. PMC: 2652338. DOI: 10.1074/jbc.M805406200. View

Goya Grocin A, Serwa R, Morales Sanfrutos J, Ritzefeld M, Tate E . Whole Proteome Profiling of -Myristoyltransferase Activity and Inhibition Using Sortase A. Mol Cell Proteomics. 2018; 18(1):115-126. PMC: 6317481. DOI: 10.1074/mcp.RA118.001043. View

Tan X, Wirjo A, Liu C . An enzymatic approach to the synthesis of peptide thioesters: mechanism and scope. Chembiochem. 2007; 8(13):1512-5. DOI: 10.1002/cbic.200700284. View

Frankel B, Kruger R, Robinson D, Kelleher N, McCafferty D . Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry. 2005; 44(33):11188-200. DOI: 10.1021/bi050141j. View

10.

Williamson D, Webb M, Turnbull W . Depsipeptide substrates for sortase-mediated N-terminal protein ligation. Nat Protoc. 2014; 9(2):253-62. DOI: 10.1038/nprot.2014.003. View

11.

Hemu X, El Sahili A, Hu S, Wong K, Chen Y, Wong Y . Structural determinants for peptide-bond formation by asparaginyl ligases. Proc Natl Acad Sci U S A. 2019; 116(24):11737-11746. PMC: 6576118. DOI: 10.1073/pnas.1818568116. View

12.

Jiang R, Weingart J, Zhang H, Ma Y, Sun X . End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation. Bioconjug Chem. 2012; 23(3):643-9. PMC: 3310247. DOI: 10.1021/bc200661w. View

13.

Dall E, Brandstetter H . Activation of legumain involves proteolytic and conformational events, resulting in a context- and substrate-dependent activity profile. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012; 68(Pt 1):24-31. PMC: 3253828. DOI: 10.1107/S1744309111048020. View

14.

Piper I, Struyvenberg S, Valgardson J, Johnson D, Gao M, Johnston K . Sequence variation in the β7-β8 loop of bacterial class A sortase enzymes alters substrate selectivity. J Biol Chem. 2021; 297(2):100981. PMC: 8361268. DOI: 10.1016/j.jbc.2021.100981. View

15.

Popp M, Dougan S, Chuang T, Spooner E, Ploegh H . Sortase-catalyzed transformations that improve the properties of cytokines. Proc Natl Acad Sci U S A. 2011; 108(8):3169-74. PMC: 3044387. DOI: 10.1073/pnas.1016863108. View

16.

Ritzefeld M . Sortagging: a robust and efficient chemoenzymatic ligation strategy. Chemistry. 2014; 20(28):8516-29. DOI: 10.1002/chem.201402072. View

17.

Deng Y, Wu T, Wang M, Shi S, Yuan G, Li X . Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level. Nat Commun. 2019; 10(1):2775. PMC: 6591319. DOI: 10.1038/s41467-019-10696-x. View

18.

Wu Z, Guo X, Guo Z . Chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides. Chem Commun (Camb). 2010; 46(31):5773-4. PMC: 3166627. DOI: 10.1039/c0cc00828a. View

19.

Bandyopadhyay A, Cambray S, Gao J . Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation. Chem Sci. 2017; 7(7):4589-4593. PMC: 5201210. DOI: 10.1039/C6SC00172F. View

20.

Rehm F, Harmand T, Yap K, Durek T, Craik D, Ploegh H . Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase. J Am Chem Soc. 2019; 141(43):17388-17393. PMC: 7372569. DOI: 10.1021/jacs.9b09166. View