Lysine-cysteine NOS bonds

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A survey of the data in the Protein Data Bank revealed that the NOS bond likely exists "in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots."[5] They found that about 100-150 or ~0.3% of entries in the PDB with resolutions of 2.0 Å or better have Lys-Cys NOS bonds[7]. Because the NOS bond was unknown before 2019, it was often overlooked in earlier interpretations of electron density maps.[5] Three examples are given illustrating putative NOS bonds in active sites of enzymes which, if correct, were previously overlooked:

  • 1m3q 2004, a DNA glycosylase: Lys249 – Cys253.
  • 5y72 2018, a prenyltransferase: Lys275 – Cys223.
  • 6t3x 2020, a cytomegalovirus nuclear egress protein: Lys132 – Cys54.


This is a summary of the observations by Wensien et al. (2021) supporting the NOS bond[5]. 6zx4 (oxidized form, NOS present, enzyme inactive) has a resolution of 0.96 Å, with a better than average Rfree of 0.136. Neisseria gonorrhoeae transaldolase has 3 cysteines (no disulfide bonds). It does not form disulfide-linked oligomers. Each Cys was individually mutated to Ser. Only the mutation Cys38Ser abolished redox control, producing a constitutively active enzyme.

Electron density for an unidentified atom appeared between the sidechain nitrogen of Lys8 and the sulfur of Cys38 for several crystals under nonreducing conditions, as well as in data from a low-dose non-synchrotron source, arguing against a radiation damage artifact. Competitive refinements indicated that the unidentified atom was oxygen, rather than carbon. Mass spectrometry analysis was consistent with this conclusion.

In crystals of reduced (active) enzyme, the Lys8-O-Cys38 bridge was absent. However, electron density near the Cys38 sulfur was consistent with molecular oxygen O2. Molecular oxygen was absent in this position in the oxidized (inactive) enzyme.

Detection and Visualization

FirstGlance in Jmol automatically detects Lys-Cys NOS bonds and alerts you to their presence. For example, take a look at 6zx4 in FirstGlance. It lists crosslinks and with one click on each, zooms in to show you the crosslink in atomic detail. Viewing the electron density map for the crosslink is just one more click. See the practical guide FirstGlance/Evaluating Protein Crosslinks. FirstGlance also alerts you a number of other kinds of protein crosslinks.

See Also

  • Electron density maps shows density maps for the bona fide NOS bond in 6zx4, as well as a presumably overlooked NOS bond in a structure published in 2011, and a case of Lys close to Cys where the electron density rules out an NOS bond.

Other Protein Crosslinks

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


  1. Ruszkowski M, Dauter Z. On methylene-bridged cysteine and lysine residues in proteins. Protein Sci. 2016 Sep;25(9):1734-6. doi: 10.1002/pro.2958. Epub 2016 Jun 17. PMID:27261771 doi:
  2. 2.0 2.1 Matthews BW. Recognizing lysine-cysteine crosslinks in proteins. Protein Sci. 2021 Aug;30(8):1491-1492. doi: 10.1002/pro.4149. Epub 2021 Jul 5. PMID:34180568 doi:
  3. Wang J. Crystallographic identification of spontaneous oxidation intermediates and products of protein sulfhydryl groups. Protein Sci. 2019 Mar;28(3):472-477. doi: 10.1002/pro.3568. Epub 2019 Jan 11. PMID:30592103 doi:
  4. Ruszkowski M, Dauter Z. Comment on Wang's paper on the covalent Cys-X-Lys bridges. Protein Sci. 2019 Mar;28(3):470-471. doi: 10.1002/pro.3576. PMID:30666728 doi:
  5. 5.0 5.1 5.2 5.3 5.4 5.5 Wensien M, von Pappenheim FR, Funk LM, Kloskowski P, Curth U, Diederichsen U, Uranga J, Ye J, Fang P, Pan KT, Urlaub H, Mata RA, Sautner V, Tittmann K. A lysine-cysteine redox switch with an NOS bridge regulates enzyme function. Nature. 2021 May 5. pii: 10.1038/s41586-021-03513-3. doi:, 10.1038/s41586-021-03513-3. PMID:33953398 doi:
  6. Fass D, Semenov SN. Previously unknown type of protein crosslink discovered. Nature. 2021 May;593(7859):343-344. doi: 10.1038/d41586-021-01135-3. PMID:33953388 doi:
  7. NOS and SONOS redox switches in proteins, von Pappenheim, Wensien, Ye, Uranga, Funk, Mata & Tittmann, preprint posted July, 2021. DOI 10.21203/

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