Isopeptide bond

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An isopeptide bond is an amide bond between the sidechain of one amino acid and the sidechain or main chain terminus of another amino acid, or a ligand. Isopeptide bonds are sometimes called lactams[1][2].

Isopeptide bonds can form covalent links between polypeptide backbones. This is in contrast to the much more common peptide (eupeptide) bonds between amino acid main chain atoms that form the polypeptide backbones of proteins. Isopepide bonds are rare (as are other non-disulfide protein crosslinks). Disulfide bonds are a much more common form of covalent linkage between polypeptide chains.

For more, please see Isopeptide bond in Wikipedia.

Contents

Formation

Spontaneous

Some isopeptide bonds appear to form spontaneously during protein folding. These include

  • Intramolecular (within molecule) isopeptide bonds in pilin monomers[3][4][5][6].
  • Intermolecular (between molecule) "chain mail" isopeptide bonds in the capsid of bacteriophage HK97[7].
  • Engineered intramolecular isopeptide bonds between collagen mimetic peptides[8].

Enzymatic

  • Poly-ubiquitins[9].
  • Intermolecular (between molecule) isopeptide bonds that link bacterial pilin monomers into long chains of pili are formed enzymatically by a cysteine transpeptidase "sortase"[3][4]. However, the same pilins have multiple intramolecular (within molecule) isopeptide bonds that appear to form spontaneously[3][4].

Cleavage

Isopeptidases are enzymes that cleave isopeptide bonds. There are many, including, for a few examples:

  • Sentrin/SUMO-specific proteases (SENP) have isopeptidase activity that can deconjugate SUMO (small ubiquitin-like modifier) from its conjugates[10][11].
  • De-ubiquitinases [12][13][14].

Frequency

A search for isopeptide bond in the keywords records at the PDB returns about 200 hits (April, 2021). This suggests that approximately one model per thousand in the PDB contains isopeptide bonds. However, not all entries containing the term isopeptide actually have isopeptide bonds in the asymmetric unit, and some entries that do have isopeptide bonds do not contain the word "isopeptide" anywhere in their PDB files.

PDB KEYWDS, REMARK 500, and LINK Records

Structures containing isopeptide bonds sometimes have "ISOPEPTIDE BOND" in their PDB file KEYWDS record. More importantly, many list isopeptide bonds in detail in either of the following two records:

Isopeptide Bond Specifications
PDB Format mmCIF Format
REMARK 500 _pdbx_validate_close_contact
LINK _struct_conn

In July, 2021, use of these records is inconsistent. Sometimes one is used, sometimes the other, sometimes some isopeptide bonds are in REMARK 500 CLOSE CONTACTS and other isopeptide bonds in the same model are in LINK records (5i6a), and sometimes isopeptide bonds that are present are not documented in either kind of record (e.g. 1fma in July, 2021). In 2021, the wwPDB is considering remediation to properly annotate all isopeptide bonds[15]. See example PDB IDs below.

Here is an example of the relevant REMARK 500 records from 2xi9. The lines with ATM1=NZ are isopeptide bonds; those with ATM1=SG are thioester bonds.
REMARK 500 THE FOLLOWING ATOMS ARE IN CLOSE CONTACT.                            
REMARK 500                                                                      
REMARK 500  ATM1  RES C  SSEQI   ATM2  RES C  SSEQI           DISTANCE          
REMARK 500   NZ   LYS B   297     CG   ASP B   595              1.31     
REMARK 500   NZ   LYS A   297     CG   ASP A   595              1.32     
REMARK 500   SG   CYS B   426     CD   GLN B   575              1.63     
REMARK 500   SG   CYS A   426     CD   GLN A   575              1.64
Here are the relevant LINK records from 3htl:
LINK         NZ  LYS X 199                 CG  ASN X 321     1555   1555  1.43  
LINK         NZ  LYS X 363                 CG  ASN X 482     1555   1555  1.55

Note that link records involving MSE (selenomethionine) generally signify nothing more than MSE being part of a polypeptide chain, but are required because the HETATM MSE residue is covalently linked to the adjacent standard amino acids. Similarly, all covalent connections to D-amino acids 5i6a and ligands should be listed in LINK records.

Examples

Journal references will be found by clicking the 4-character PDB codes.

Specified

Isopeptide bonds in the following examples are specified in LINK/_struct_conn records.

  • The capsid of the HK97 bacteriophage contains isopeptide bonds that cross-link the protein chains into a protein chain mail [16][17][7]. However, these do not occur in the asymmetric unit of 1ohg[18]. They can be visualized in the biological unit, which is the full virus capsid, but that is technically challenging.
  • 7cap is a ubiquitin homotrimer. 3ALB is a ubiquitin homotetramer. In both cases, the C-terminal Gly76 is isopeptide linked to Lys48 in each inter-chain interface[9].
  • 4mli contains the SpyTag peptide (which can be linked to a protein of interest) forming an isopeptide bond between SpyTag peptide Lys31 and SpyCatcher protein Asp117[19].
  • 3htl is a pilus monomer with spontaneously-formed intra-chain isopeptide bonds Lys199-Asn321 and Lys363-Asn482[20]. (Pilin chains form enzymatically, linked by the isopeptide Lys169 sidechain bonded to the carboxy terminus Thr494. Those inter-chain isopeptide bonds are not present in the monomer structure.)
  • 6vzx: triple helix formed from collagen-mimetic peptides, stabilized by Lys11-Glu13 inter-chain isopeptide bonds.
  • 5i6a: 10-amino acid peptides with both N-terminal and C-terminal isopeptide bonds, some involving D-amino acids.

Unspecified

Isopeptide bonds in the following examples were specified in neither REMARK 500 CLOSE CONTACTS/_pdbx_validate_close_contact nor LINK/_struct_conn records in May, 2021[15]. Subsequently, after being reported to the wwPDB, these may have been remediated, which will be indicated in REVDAT/_database_PDB_rev records.

  • 1fma: Molybdopterin synthase, an enzyme with structural similarity to ubiquitin, has Lys118 chain E isopeptide bonded to the carboxy-terminal Gly81 in chain D.
  • 5jqf: Lasso peptide sphingopyxin I has Asp9 isopeptide bonded to the amino terminus at Gly1. This bond is cleaved by lasso peptide isopeptidase.

Pyroglutamate

Pyroglutamate with electron density map
Pyroglutamate with electron density map

N-terminal Glu or Gln can cyclize, forming an internal lactam (isopeptide) bond. See Pyroglutamic acid in Wikipedia. Examples with convincing electron densities include 1s1a (PCA1) and 2are (Gln1). Element color key: C O N.

Detection and Visualization

FirstGlance in Jmol alerts you to isopeptide bonds when present, and provides convenient links to that zoom and and display each one in detail. Viewing the electron density map is just one more click. Use the links above under Examples to go to a Proteopedia page titled with a 4-character PDB code. There, click on "FirstGlance". In FirstGlance, click on the Tools tab, and there, on "Protein Crosslinks". See the practical guide FirstGlance/Evaluating Protein Crosslinks.

Other Types of Protein Crosslinks

In addition to the thioester bonds discussed above, other covalent cross-links between polypeptide chains include:

References

  1. ↑ Hegemann JD, Zimmermann M, Zhu S, Steuber H, Harms K, Xie X, Marahiel MA. Xanthomonins I-III: A New Class of Lasso Peptides with a Seven-Residue Macrolactam Ring. Angew Chem Int Ed Engl. 2014 Jan 20. doi: 10.1002/anie.201309267. PMID:24446383 doi:http://dx.doi.org/10.1002/anie.201309267
  2. ↑ Ma Y, Yue Y, Ma Y, Zhang Q, Zhou Q, Song Y, Shen Y, Li X, Ma X, Li C, Hanson MA, Han GW, Sickmier EA, Swaminath G, Zhao S, Stevens RC, Hu LA, Zhong W, Zhang M, Xu F. Structural Basis for Apelin Control of the Human Apelin Receptor. Structure. 2017 Jun 6;25(6):858-866.e4. doi: 10.1016/j.str.2017.04.008. Epub 2017, May 18. PMID:28528775 doi:http://dx.doi.org/10.1016/j.str.2017.04.008
  3. ↑ 3.0 3.1 3.2 Nakata M, Kreikemeyer B. Genetics, Structure, and Function of Group A Streptococcal Pili. Front Microbiol. 2021 Feb 9;12:616508. doi: 10.3389/fmicb.2021.616508., eCollection 2021. PMID:33633705 doi:http://dx.doi.org/10.3389/fmicb.2021.616508
  4. ↑ 4.0 4.1 4.2 Kang HJ, Baker EN. Intramolecular isopeptide bonds give thermodynamic and proteolytic stability to the major pilin protein of Streptococcus pyogenes. J Biol Chem. 2009 Jul 31;284(31):20729-37. doi: 10.1074/jbc.M109.014514. Epub, 2009 Jun 4. PMID:19497855 doi:http://dx.doi.org/10.1074/jbc.M109.014514
  5. ↑ Kang HJ, Baker EN. Intramolecular isopeptide bonds: protein crosslinks built for stress? Trends Biochem Sci. 2011 Apr;36(4):229-37. doi: 10.1016/j.tibs.2010.09.007. Epub , 2010 Nov 4. PMID:21055949 doi:http://dx.doi.org/10.1016/j.tibs.2010.09.007
  6. ↑ Baker EN, Squire CJ, Young PG. Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria. Biochem Soc Trans. 2015 Oct;43(5):787-94. doi: 10.1042/BST20150066. PMID:26517883 doi:http://dx.doi.org/10.1042/BST20150066
  7. ↑ 7.0 7.1 Dierkes LE, Peebles CL, Firek BA, Hendrix RW, Duda RL. Mutational analysis of a conserved glutamic acid required for self-catalyzed cross-linking of bacteriophage HK97 capsids. J Virol. 2009 Mar;83(5):2088-98. doi: 10.1128/JVI.02000-08. Epub 2008 Dec 17. PMID:19091865 doi:10.1128/JVI.02000-08
  8. ↑ Hulgan SAH, Jalan AA, Li IC, Walker DR, Miller MD, Kosgei AJ, Xu W, Phillips GN Jr, Hartgerink JD. Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs. Biomacromolecules. 2020 Aug 21. doi: 10.1021/acs.biomac.0c00878. PMID:32820897 doi:http://dx.doi.org/10.1021/acs.biomac.0c00878
  9. ↑ 9.0 9.1 Hiranyakorn M, Yanaka S, Satoh T, Wilasri T, Jityuti B, Yagi-Utsumi M, Kato K. NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains. Int J Mol Sci. 2020 Jul 28;21(15). pii: ijms21155351. doi: 10.3390/ijms21155351. PMID:32731397 doi:http://dx.doi.org/10.3390/ijms21155351
  10. ↑ Liu Y, Shen Y, Song Y, Xu L, P Perry JJ, Liao J. Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach. Biomolecules. 2021 Apr 30;11(5). pii: biom11050673. doi: 10.3390/biom11050673. PMID:33946350 doi:http://dx.doi.org/10.3390/biom11050673
  11. ↑ Tokarz P, Wozniak K. SENP Proteases as Potential Targets for Cancer Therapy. Cancers (Basel). 2021 Apr 24;13(9). pii: cancers13092059. doi:, 10.3390/cancers13092059. PMID:33923236 doi:http://dx.doi.org/10.3390/cancers13092059
  12. ↑ Majumdar P, Nath U. De-ubiquitinases on the move: an emerging field in plant biology. Plant Biol (Stuttg). 2020 Jul;22(4):563-572. doi: 10.1111/plb.13118. Epub 2020, Apr 30. PMID:32233097 doi:http://dx.doi.org/10.1111/plb.13118
  13. ↑ Yang XD, Sun SC. Deubiquitinases as pivotal regulators of T cell functions. Front Med. 2018 Aug;12(4):451-462. doi: 10.1007/s11684-018-0651-y. Epub 2018 Jul , 27. PMID:30054854 doi:http://dx.doi.org/10.1007/s11684-018-0651-y
  14. ↑ Sahtoe DD, Sixma TK. Layers of DUB regulation. Trends Biochem Sci. 2015 Aug;40(8):456-67. doi: 10.1016/j.tibs.2015.05.002. Epub , 2015 Jun 11. PMID:26073511 doi:http://dx.doi.org/10.1016/j.tibs.2015.05.002
  15. ↑ 15.0 15.1 Remediation of the PDB database to guarantee proper annotation of all isopeptide bonds was discussed with Rachel Kramer Green of the RCSB in June, 2021.
  16. ↑ Hendrix RW, Johnson JE. Bacteriophage HK97 capsid assembly and maturation. Adv Exp Med Biol. 2012;726:351-63. doi: 10.1007/978-1-4614-0980-9_15. PMID:22297521 doi:10.1007/978-1-4614-0980-9_15
  17. ↑ Gilakjan ZA, Kropinski AM. Cloning and analysis of the capsid morphogenesis genes of Pseudomonas aeruginosa bacteriophage D3: another example of protein chain mail? J Bacteriol. 1999 Dec;181(23):7221-7. PMID:10572124
  18. ↑ Wikoff WR, Liljas L, Duda RL, Tsuruta H, Hendrix RW, Johnson JE. Topologically linked protein rings in the bacteriophage HK97 capsid. Science. 2000 Sep 22;289(5487):2129-33. PMID:11000116
  19. ↑ Li L, Fierer JO, Rapoport TA, Howarth M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J Mol Biol. 2013 Oct 23. pii: S0022-2836(13)00666-9. doi:, 10.1016/j.jmb.2013.10.021. PMID:24161952 doi:http://dx.doi.org/10.1016/j.jmb.2013.10.021
  20. ↑ Kang HJ, Paterson NG, Gaspar AH, Ton-That H, Baker EN. The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds. Proc Natl Acad Sci U S A. 2009 Oct 6;106(40):16967-71. Epub 2009 Sep 21. PMID:19805181

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