Isopeptide Ligation bySortase-Mediated Ligation
M1 Xiaoyi Pan240509

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Content• Introduction• Isopeptide ligation by SML• Enzymatic approach to isopeptide ligation• Acyl acceptor altering approach to isopeptide ligation-- CdSrtA-- Unnatural Amino Acid

• Summary2

Enzymatic Protein Conjugation✓Mild conditions✓High yield✓Fast kinetics

SortaseSubtiligase

Microbial
Transglutaminase(MTG)
Zhang, Y.; Distefano, M. D.; et al. Chem. Soc. Rev., 2018,47, 9106-9136
Morgan, H. E.; Turnbull, W. B.; Webb, M. E.; et al. Chem. Soc. Rev., 2022, 51, 4121-4145

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Sortase-Mediated Ligation (SML)✓High Site-Specificity✓High level of overexpression efficiency

Improvement:• Enhancing activity/robustness• Reprogramming selectivity→• reducing reversibility• etc.

Zou, Z; Ji, Y; Schwaneberg, U.; Angew. Chem. Int. Ed. 2023, 63, e202310910.

A powerful tool forpeptide ligation

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Sortase• Generally found in Gram-positive bacteria.
• Sortase A (SrtA) is the most major class among 6 classes of sortase.

Signal sequenceof SrtA

Emidio, N. B.; Cheloha, R. W.. Curr Opin Chem Biol. 2024, 80, 102443
Zou, Z.; Ji, Y; Schwaneberg, U.. Angew. Chem. Int. Ed. 2023, 63, e202310910.

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SaSrtA• Found in S. aureus for the first time in 1999
• To anchor surface proteins to the bacterial cell wall
8-stranded β-barrel core shared across all sortaseclasses.
• His120, Cys184, and Arg197• β6/β7 loop & β7/β8 loop are crucial for activity andsorting specificity

Mazmanian, S. K.; Schneewind, O. et al. Sci. 1999, 285, 760-763
Zou, Z; Ji, Y; Schwaneberg, U.; Angew. Chem. Int. Ed. 2023, 63, e202310910.

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Previous application• N-terminal• Oligoglycine as acylaccepter→How about apply toisopeptide ligation?

Zou, Z; Ji, Y; Schwaneberg, U.; Angew. Chem. Int. Ed. 2023, 63, e202310910.

7Isopeptide Ligation• Side reaction

Conjugate with
Lys side chain

×Low selectivity
×Inefficient

Möhlmann, S.; Harrenga, A. et al. ChemBioChem, 2011, 12, 1774-1780
Dasgupta, S.; Roy, R. P. et al. JBC, 2011, 286, 23996-24006

8Content• Introduction• Isopeptide ligation by SML• Enzymatic approach to isopeptide ligation• Acyl acceptor altering approach to isopeptide ligation-- CdSrtA-- Unnatural Amino Acid

• Summary

9SrtC• Class C: Pilins biosynthesis related• Signal sequence: LPLTG
pilin subunit

Bradshaw, W. J.; Acharya, K. R.; et al. FEBS J, 2015, 282, 2097-2114

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Structure of SrtC
• Lid

• Responsible for recognizing signal sequence and pilin motif.

Spirig, T.; Weiner, E.M.; Clubb, R.T. Mol. Microbiol. 2011, 82, 1044-1059

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CdSrtA• From Corynebacterium diphtheriae• H160、C222、R231

pilin subunit

McConnell, S. A.; Clubb, R. T. et al. J. Am. Chem. Soc. 2018, 140, 8420–8423

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CdSrtA3M• Mutation: D81G, W83G, N85A
0 24h 48h

0 24h 48h

✓In vitro

McConnell, S. A.; Clubb, R. T. et al. J. Am. Chem. Soc. 2018, 140, 8420–8423

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CdSrtAΔ

Sue, C. K.; Clubb, R. T. et al. Bioconjugate Chem. 2020, 31, 1624–1634

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Dual labeling

24h

McConnell, S. A.; Clubb, R. T. et al. J. Am. Chem. Soc. 2018, 140, 8420–8423

2h15

Short Summary✓Mild condition✓High selectivity

Kinetics of CdSrtA Catalyzed Lysine–Isopeptide Formation

k cat ×10–5 (s–1)

NK

M ×
10–4 (M)SK

M ×
10–4 (M)k cat/NKM

CdSrtA

n.d.

n.d.

n.d.

n.d.

CdSrtA3M

5.6 ± 0.8

0.7 ± 0.1 20 ± 10

0.7 ± 0.1

CdSrtAΔ

40 ± 0.1

1.6 ± 0.4 16 ± 3

2.5 ± 0.6

SaSrtA

1600 ± 100 1.8 ± 0.1 73.3 ± 10.1 86 ± 5(s–1 M–1)

×Slow×Pilin Motif requiredSue, C. K.; Clubb, R. T. et al. Bioconjugate Chem. 2020, 31, 1624–1634

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Unnatural Amino AcidFottner, M.; Lang, K. et al. Nat. Chem. Biol. 2019, 15, 276-284

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In vitro

Fottner, M.; Lang, K. et al. Nat. Chem. Biol. 2019, 15, 276-284
Kulathu, Y.; Komander, D. Nat Rev Mol Cell Biol, 2012, 13, 508–523

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in cellulo• Applicable for complex and multidomain
Fottner, M.; Lang, K. et al. Nat. Chem. Biol. 2019, 15, 276-284

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Resistant to DUB (deubiquitylase)

Fottner, M.; Lang, K. et al. Nat. Chem. Biol. 2019, 15, 276-284

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Structurally Equivalent to WT• Able to be recognized by antibody of WT
Fottner, M.; Lang, K. et al. Nat. Chem. Biol. 2019, 15, 276-284

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Type of SrtA

✓Orthogonality of 2 types Sortase

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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Structure of ubiquitylation

Kulathu, Y.; Komander, D. Nat Rev Mol Cell Biol, 2012, 13, 508–523

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Ubiquitylation bySrtA

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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Study of SUMO binding site

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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Study of SUMO binding site

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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Study of SUMO binding site

• K21-linked diUb-SUMO2 hybrid chain
Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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Summary• Sortase✓Mild Condition✓High Site Specificity✓Fast Kinetics✓High yield✓High Overexpression efficiency• Generally, linking N-terminal to oligoglycine

28Summary
Enzymatic Approach

Acceptor altering approach

Using Sortase C as enzyme✓In vitro✓Dual labelingUsing unnatural amino acid asacyl acceptor✓in vitro ✓in cellulo✓Dual labeling

×Pilin motif required
×Slow kinetics✓Applicable for complex andmultidomain✓Resistibility to DUB✓Structurally Equivalent to WT
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Thanks for Listening

30Appendix31

6 classes of sortase
SrtA:

Ca2+ needed
Recognized sequence: LPXTG (CWSS)SrtB:Free iron needed
Recognized sequence: NPQTN
Bradshaw, W. J.; Acharya, K. R.; et al. FEBS J, 2015, 282, 2097-2114

SrtC:

Pilus-specific enzyme
Recognized sequence: LPLTG
SrtD:

Related to sporulating32

6 classes of sortase

SrtE: Housekeeping Sortase
Recognized sequence: LAXTG

Bradshaw, W. J.; Acharya, K. R.; et al. FEBS J, 2015, 282, 2097-2114
Girolamo, S. D.; Lipps, G.; et al. Biochem J, 2019, 476, 665-682

SrtF: From P. acnes
No need of Ca
Recognized sequence: LPXTG

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Reaction mechanism of SaSrtA

Bradshaw, W. J.; Acharya, K. R.; et al. FEBS J, 2015, 282, 2097-2114

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GG-motif• Diglycine is necessary for nucleophile of SaSrtA

Huang, X.; Ellestad, G. et al. Biochemistry, 2003, 42, 11307-11315

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Signal sequence of CdSrtA3M & CdSrtΔ

Sue, C. K.; Clubb, R. T. et al. Bioconjugate Chem. 2020, 31, 1624–1634

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Pilin motif

McConnell, S. A.; Clubb, R. T. et al. J. Am. Chem. Soc. 2018, 140, 8420–8423

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2DPBA

Luo, J; Deiters, A.; et al. Nature Chem, 2016, 8, 1027-1034

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Srt5M• P94R/D160N/D165A/K190E/K196T

k cat、s -1
WT(SaSrtA)

1.5±0.2

KmLPETG 、mM

P94R/D160N/D165A/K
190E/K196T (5M)5.4±0.40.23±0.02

7.6±0.5

Chen, I.; Dorr, B. M.; Liu, D. R. PNAS, 2011, 108, 11399-11404

k cat / K m LPETG、M -1 s -1 K m GGG-COOH、μM
200±30140±3023,000±3,000

2,900±200

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Srt5M & Srt2A

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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NMR of K21

Fottner, M.; Lang, K. et al. Nat. Commun. 2021, 12, 6515

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