Lysine-cysteine NOS bonds

Covalent Lysine-Cysteine protein crosslinks were first reported in 2016, interpreted at the time as Lys-CH2-Cys. In 2019, Jimin Wang provided evidence for oxygen rather than methylene as the linker. In 2021, Wensien et al. with the Tittmann group (Goettingen), studying Transaldolase, put the Lysine-cysteine "Nitrogen-Oxygen-Sulfur" (NOS) protein crosslink on firm ground (). The sidechains of a lysine and a cysteine, joined by an NOS bond, make a covalent linkage between amino acids in a polypeptide chain (or chains). For comparison, the disulfide bond is a far more common type of covalent linkage between polypeptide chains, and the isopeptide bond is another rare type. near the N-terminus of the 352 amino acid Neisseria gonorrhoeae transaldolase chain 6zx4, between Lys8 and Cys38, near the surface.  Amino Terminus                 Carboxy Terminus  Reduction breaks the NOS bond. In transaldolase, breaking the NOS bond causes subtle allosteric shifts in the catalytic site, increasing enzymatic activity by several orders of magnitude. Thus, the NOS bond is described as an allosteric redox switch. Reduced: NOS bond broken, enzyme active. Oxidized: NOS bond present, enzyme inactive.

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PrevalencePrevalence

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." 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. Because the NOS bond was unknown before 2019, it was often overlooked in earlier interpretations of electron density maps. 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.

MethodsMethods

This is a summary of the observations by Wensien et al. (2021) supporting the NOS bond. 6zx4 (oxidized form, NOS present, enzyme inactive) has a resolution of 0.96 Å, with a better than average R_free 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 VisualizationDetection 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 AlsoSee 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 CrosslinksOther Protein Crosslinks

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

• Disulfide bonds
• Isopeptide bonds
• Thioester protein crosslinks
• Thioether protein crosslinks
• Ester protein crosslinks
• Histidine-tyrosine protein crosslinks