Sortase A

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Sortase A
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EC no. 3.4.22.70
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Sortase A (EC 3.4.22.70, SrtA, SrtA protein, SrtA sortase) is an enzyme. [1] [2] [3] This enzyme catalyses a cell wall sorting reaction, in which a surface protein with a sorting signal containing a LPXTG motif, is cleaved between the Thr and Gly residue.

Contents

This enzyme belongs to the peptidase family C60.

Structure

Sortase A has an eight stranded β-barrel fold with a hydrophobic cleft formed by β7-β8 strands. This cleft is surrounded by β3-β4, β2-β3, β6-β7, and β7-β8 loops. The catalytic cysteine residue is found in this cleft and accepts subsequent binding of a nucleophilic agent. The β3-β4 loop contains a calcium binding site which binds calcium via coordination to a residue in the β6-β7 loop. Such binding slows down the motion of the β6-β7 loop, allowing the substrate of Sortase to bind and increase its activity eightfold. [4]

Use in protein engineering

Sortase A has been widely used as an in vitro tool to post-translationally modify proteins at the N- and C-termini with an appended label. These labels include biotin, fluorophores, crosslinkers, and multifunctional probes. [5]

In both cases, one molecule is engineered to contain a LPXTG motif at one end and another molecule is engineered to contain a (Gly)n motif at another end. Upon cleavage of the LPXTG motif, Sortase forms a thioester intermediate with the engineered molecule. This intermediate is then resolved by nucleophilic attack by the (Gly)n containing molecule to form a fusion between the two molecules with an intervening LPXT(Gly)n motif.

To achieve N-terminal labeling of a protein, the LPXTG motif is engineered to be at the C-terminus of the label. The protein is engineered to have an N-terminal (Gly)n. To achieve C-terminal labeling of the same protein, the LPXTG motif is engineered to be at the C-terminus of the protein. A (Gly)n molecule is engineered to contain the label at its C-terminus.

Finally, both N and C-termini of proteins can be labeled by using Sortases of different substrate specificity. For example, Sortase A from streptococcus pyogenes, recognizes and cleaves the LPXTA motif and accepts Ala-based nucleophiles. This SrtA also recognizes and cleaves the LPXTG motif with reduced efficiency. However, Staph. A. Sortase A does not recognize LPXTA substrates and thus are orthogonal to the LPXTA sequence.

In addition, Sortase A has also been used to piecewise create proteins, protein domains, and peptides. [6]

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References

  1. ↑ Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O (October 1999). "Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif". Proceedings of the National Academy of Sciences of the United States of America. 96 (22): 12424–9. Bibcode:1999PNAS...9612424T. doi: 10.1073/pnas.96.22.12424 . PMC   22937 . PMID   10535938.
  2. ↑ Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV (July 2004). "Crystal structures of Staphylococcus aureus sortase A and its substrate complex". The Journal of Biological Chemistry. 279 (30): 31383–9. doi: 10.1074/jbc.m401374200 . PMID   15117963.
  3. ↑ Race PR, Bentley ML, Melvin JA, Crow A, Hughes RK, Smith WD, Sessions RB, Kehoe MA, McCafferty DG, Banfield MJ (March 2009). "Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism". The Journal of Biological Chemistry. 284 (11): 6924–33. doi: 10.1074/jbc.m805406200 . PMC   2652338 . PMID   19129180.
  4. ↑ Suree N, Liew CK, Villareal VA, Thieu W, Fadeev EA, Clemens JJ, Jung ME, Clubb RT (September 2009). "The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized". The Journal of Biological Chemistry. 284 (36): 24465–77. doi: 10.1074/jbc.M109.022624 . PMC   2782039 . PMID   19592495.
  5. ↑ Popp MW, Antos JM, Ploegh HL (April 2009). "Site-specific protein labeling via sortase-mediated transpeptidation". Current Protocols in Protein Science. Chapter 15: 15.3.1–15.3.9. doi:10.1002/0471140864.ps1503s56. PMC   5551486 . PMID   19365788.
  6. ↑ Popp MW, Ploegh HL (May 2011). "Making and breaking peptide bonds: protein engineering using sortase". Angewandte Chemie. 50 (22): 5024–32. doi:10.1002/anie.201008267. PMID   21538739.
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