NOS and SONOS redox switches in proteins

We recently reported on the discovery of a lysine-cysteine redox switch in proteins with a covalent NOS bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous, hitherto overlooked redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines forming a SONOS bridge with a trivalent nitrogen, which constitutes the first native branching crosslink in proteins. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors linking lysine chemistry and redox biology


Introduction
Reactive oxygen species (ROS) are central to redox signaling in all domains of life and critically control cell growth, development, metabolism, aging and the response to stress conditions as e.g.infection by pathogens [1][2][3][4] .At elevated levels, ROS induce oxidative stress that has been implicated in a myriad of pathologies including cancer, neurodegenerative diseases, inflammation and autoimmune conditions 5,6 .
The underlying molecular mechanisms of redox signaling and oxidative stress have been mostly attributed to chemical modifications of cysteine residues in redoxsensitive proteins with key reactions being disulfide bridge formation between two cysteines or oxidation, glutathionylation and nitrosylation of individual cysteines 7,8 .
We have recently reported the discovery of an allosteric lysine-cysteine redox switch with a covalent NOS bridge that regulates the enzymatic activity in response to changing redox conditions 9 .Here, we systematically mine the available protein structure database for proteins with undetected lysine-cysteine redox switches and find hitherto unidentified NOS bridges in proteins from all domains of life with critical roles in central cellular functions including metabolism, gene expression, signaling, the ubiquitin pathway, DNA repair and redox homeostasis.These findings bear wide-ranging biological implications in the context of redox signaling, oxidative stress and many human disease states.

Geometric properties of NOS bridges
Before we analyzed the deposited experimental protein structures in the protein database (www.rcsb.org) to search for potentially undetected covalent NOS bridges, we set out to define the geometric properties of these versus non-covalent H-bond interactions between cysteine and lysine residues to identify reliable criteria to discern between these scenarios.A survey of H-bond interactions of cysteines in protein structures disclosed mean interatomic S-N distances of 3.44 Å with lysine residues (cysteine as acceptor) and 3.75 Å for the case when cysteine acts as Hbond donor 10 .However, the closest possible physical encounter between the two side chains remained to be defined.To close this gap of knowledge, we conducted quantum chemical calculations, where we analyzed the geometric properties of i) the NOS bridge, ii) a hydrogen bond between lysine as amine (-NH2) and cysteine (-SH) and iii) a hydrogen bond between protonated lysine (-NH3 + ) and cysteine (-SH).An extensive conformational search was performed for the two residues in all three scenarios at different α-carbon atom distances (ED Fig. 1).Several theoretical methods (also considering different dielectric constants, SI Fig. 1) provided a consistent picture, whereby all minima featuring an N-S distance below 3.1 Å exhibited a covalent NOS bridge.The covalently bound NOS conformers displayed a bimodal distribution of the N-S 1,3 distance, with two maxima in the 2.6-2.7 Å range.This is a purely steric effect resulting from the relative positioning of the sulfur atom to the lysine chain (SI Fig. 2).Hydrogen-bonded lysine-cysteine pairs are formed at much larger distances, with minimal values hovering at 3.2 Å for a protonated lysine-cysteine interaction, increasing to 3.5 Å once a proton is removed.
The computational results signal a clear difference between the three types of interactions, with the lowest energy distributions showing no overlap.The results are also consistent independent of the relative positioning of the two residues.

Detectability of NOS bridges in crystallographic protein structures
Next, we tested the impact of resolution and deposited dose in X-ray crystallographic experiments on the traceability of covalent NOS bridges in protein structures (ED Fig. 2).We had initially detected the NOS redox switch in protein crystals that diffracted beyond 1 Å, a resolution that is rarely accomplished in protein crystallography but is required to unambiguously define the chemical nature of atoms and their connectivities 11 .We used a sub-ångstrøm dataset obtained for one of these crystals and truncated the diffraction data at 1.0, 1.5, 2.0, 2.5 and 3.0 Å, and further omitted the bridging oxygen atom of the NOS bridge for structure refinements to eliminate model bias (ED Fig. 2a).As one would expect, the detectability of the covalent crosslink is critically dependent on the resolution: while the presence of the bridging atom is verifiable up to resolutions of 2 Å based on both the 2mFo-DFc as well as mFo-DFc difference electron density maps, the interpretation of electron density maps at lower resolutions becomes ambiguous.In the transaldolase protein crystals we analyzed, the NOS bridge exhibits almost full occupancy and our system may therefore be regarded as a best-case scenario (also considering the low B-factors).To test the impact of high-energy synchrotron radiation on the NOS bridge in protein crystals, which is known to promote "radiation damage" of redox-sensitive groups 12 , we conducted dose-dependent experiments depositing calibrated doses between 0.5 and 10 MGy in 0.5 MGy increments (ED Fig. 2b, X-ray crystallographic statistics in SI Table 1).These experiments reveal that the NOS bridge in the tested protein crystals is relatively robust and withstands doses up to 5 MGy, where radiation damage at neighboring acidic side chains becomes visible.Only when we deposited high doses of 10 MGy, we detected a slight decomposition of the NOS bridge.In sum, to reliably detect NOS bridges in protein crystal structures, a resolution of better than 2 Å is required as well as a high occupancy of this group and sufficient local order."Radiation damage" can be excluded to cause NOS bridge formation artificially as we have demonstrated before 9 , to the contrary, NOS bridges decompose while interacting with high-energy radiation but only at very high doses.
On the basis of the quantum chemical calculations and the X-ray crystallographic experiments on our model system, we downloaded all pdb entries with a resolution of ≤2 Å (65 000) including the corresponding structure factors and electron density maps, if available.We then searched for close contacts between lysine and cysteine residues, specifically pairs with interatomic N-S distances of less than 3 Å, which are in the regime of covalent NOS bridges rather than H-bond interactions (>3.2 Å).
We identified 266 deposited structures with ~400 potential NOS bridges (SI Data 1), and manually inspected the putative lysine-cysteine crosslink site in all these structures.In almost all deposited structures, the corresponding lysine and cysteine residues were built without a crosslink, in very few with a methylene bridge and in one except our own structures with an NOS link.We remodeled both residues with a covalent crosslink, either as an NOS bridge, or, alternatively, as a direct N-S linkage (sulfenamide) using geometrically parametrized restraints.We consider formation of a methylene (N-CH2-S) bridge as highly unlikely as previously discussed by us and others 9,13 .We then re-refined the structures and compared the obtained models and electron density maps for the three alternative scenarios: i) a covalent NOS linkage, ii) a covalent N-S linkage and iii) a non-covalent H-bond interaction (as deposited).In ~150 data sets, the existence of an NOS bridge is highly likely (~100) or possible (~50), while a non-covalent H-bonding interaction or direct N-S linkage can be relegated in these data sets to minor probability (SI Data 1).In a few datasets, the electron density maps clearly indicate the presence of a crosslink but the quality is insufficient to discriminate between a NOS bridge and a direct N-S bond (sulfenamide).Proteins from human or plant pathogens (ED Table 1) and human proteins (ED Table 2) are summarized individually.

Structural and chemical motifs of NOS bridges
NOS bridges are observed in an amazing diversity of structural motifs and chemical variations (Fig. 1, Fig. 2).First, we analysed the distance distribution in sequence between the NOS bridge lysine and cysteine residues −Cys a −(X)n-1−Lys a+n −, where a and a+n denote the corresponding sequence positions and n the sequence distance (Fig. 2a).This analysis reveals a Gauss-like distribution with the largest fraction of NOS bridges observed for short sequence distances (n <|10|) and the smallest distance being two positions in sequence (n+2) in intra-strand/strand-like structures (Fig. 2b).Other motifs include intra-helical NOS bridges (mostly n+4) (Fig. 2c), inter-strand bridges in e.g.barrel structures (Fig. 2d), bridges connecting helices and strands (Fig. 2e), or intra-loop bridges (Fig. 2f).Most NOS bridges are formed intramolecularly within one protein chain, in a few examples we detected intermolecular crosslinks, either as single bridges between two different proteins (A-B type) or as an NOS "double bridge" in homodimeric assemblies (A2-type), where the corresponding lysine and cysteine residues of each chain form two reciprocal NOS bridges (Fig. 1, Fig. 2g).In one case, an NOS bridge is observed for a cysteine that is alternatively engaged in a disulfide bridge suggesting a "mixed" disulfide-NOS redox switch site (Fig. 2i).In terms of the oxidation state of the NOS cysteine, we exclusively found mono-oxidized species (sulfenic acid equivalent), there is not a single case where the cysteine sulfur is in a higher oxidation state (bonded to more than one oxygen atom).However, we discovered that in some proteins lysines may form NOS bridges with two cysteines at the same time constituting a branching SONOS bridge, in which the lysine nitrogen is in a di-oxidized nitro-state as shown for e.g.galectin-1, a redox-sensitive protein with numerous important biological functions 14 (Fig. 1, Fig. 2h, ED Table 2).To the best of our knowledge, the SONOS group would be the first native branching crosslink between amino acids in proteins.
Amino-acid derived redox cofactors, which are formed through crosslinks between two and in rare cases three amino acids (e.g.Met-Tyr-Trp) do not possess a single branching center as the lysine nitrogen atom of the SONOS group 15,16 .Other examples for proteins with SONOS bridges include the main protease (Mpro) from SARS-CoV-2 (Fig. 3), an important drug target for fighting the current COVID-19 pandemic 17,18 , and the human Nsf1-IscU complex that is central to the Fe-S cluster biogenesis in mitochondria 19 (ED Fig. 3).In both cases, structures with either an NOS or a SONOS bridge of the same redox switch were detected suggesting a stepwise oxidation (Fig. 1).While the initial oxidation (NOS) involved structurally proximal lysine and cysteine residues, the second oxidation (SONOS) involved in both cases a 'mobile' cysteine that structurally fluctuates between the active sites and the redox site (Fig. 3, ED Fig. 3).

Catalytic requirements for NOS/SONOS bridge formation
In our initial discovery of the allosteric NOS redox switch in the enzyme transaldolase from Neisseria gonorrhoeae 9 , we had speculated that a neighboring glutamate (Glu93) might be catalytically essential for formation of the NOS bridge akin to the suggested role of a glutamate in isopeptide bond formation in some proteins 20 (ED Fig. 4a).However, in many proteins with identified NOS or SONOS bridges, no amino acid capable of acid-base catalysis is found in the immediate vicinity of the redox switch pinpointing to an intrinsic reactivity of structurally proximal lysine and cysteine residues provided ROS and/or O2 are present.To test this hypothesis, we generated a transaldolase Glu93Gln variant, in which a potential catalytic role of residue 93 is eliminated, and analysed the structure and enzymatic properties of the variant.The kinetic constants are very similar to the wild-type enzyme except for a slight 1.5-fold increase in the substrate Michaelis constant (SI Table 2), and the variant remains redox activatable.The structural analysis of the Glu93Gln variant by X-ray crystallography (SI Table 1) clearly indicates formation of the NOS bridge argueing against a compulsory catalytic mode involving a neighboring amino acid (ED Fig. 4b).This mechanistic proposal is also supported by the finding that a lysine-cysteine crosslink was unintentionally engineered into the penicillin-binding protein 4 from Staphylococcus aureus, where the catalytic nucleophile Ser95 at the active site had been replaced by a cysteine that forms a crosslink with the co-catalytic Lys98 (ED Fig. 4c-e).Likewise, a positional inversion of the NOS-bridge forming lysine and cysteine residues in the human DNA repair enzyme OGG1 resulted in the formation of an NOS bridge akin to the wild-type configuration (ED Fig. 4f-i).While amino acid or enzyme catalyst are not compulsory for NOS bridge formation in vitro, we cannot exclude enzyme or metal ion catalysis under in vivo conditions as in case of disulfide bonds 21 .

Chemical functions of the NOS lysine and cysteine residues
In a next step, we analyzed the chemical functions of the lysine and cysteine residues forming the NOS/SONOS redox switches in the context of the respective protein structure and function.In the previously reported redox-sensitive transaldolase, the NOS bridge functions as an allosteric switch that changes the structure of the protein including that of the active site, and thereby regulates enzymatic activity 9 .The operational modes of the NOS/ SONOS redox switch residues in the now identified proteins are amazingly diverse and can be classified into several major classes including lysines with direct catalytic roles, lysines with direct binding roles, cysteines with direct catalytic roles, and allosteric switches (Fig. 4, Fig. 5).The manifold chemical tasks associated with the NOS lysine residues are particularly intriguing and include the full spectrum of lysine roles in protein function.
NOS bridges were identified for proteins with catalytic lysines that form i) Schiffbases with the PLP cofactor in PLP-dependent enzymes (e.g.arginine/ornithine decarboxylase), ii) Schiff-bases with enzymatic substrates (e.g.KDPG aldolase), iii) carbamate intermediates in covalent CO2 transfer to biotin (e.g.oxaloacetate decarboxylase/Na + pump) and lysines that iv) act as acid-base catalysts in the active site of enzymes (e.g.penicillin binding protein) (Fig. 4a-d).Apart from these catalytic roles, numerous proteins were identified, where the NOS lysine is directly involved in binding of enzymatic substrates (e.g.DAH7P synthase) or of effectors as e.g.inositolphosphates (e.g.rabphilin), often interacting with a negatively charged moiety of substrate or effector (Fig. 4e-f).In other cases, the NOS bridge is located proximal to regulatory metal binding sites (e.g.Ca 2+ ) (Fig. 4f-g).An interesting observation concerns the involvement of the NOS bridge lysines in direct binding of DNA.In e.g.DNA polymerase, the lysine interacts with the base moiety of single stranded DNA (Fig. 6a), while in homeobox protein Hox, it binds to the phosphate backbone of double stranded DNA (Fig. 6b).Interestingly, NOS redox switches are also found for histone writers and erasers suggesting a critical and multilayered role of these switches for the regulation of gene expression (Fig. 6c,d).Catalytic cysteines were detected for several ubiquitin E2 ligases (ED Fig. 5) and the FeS cluster biogenesis complex Iscu-Nsf1 (ED Fig. 3) as discussed before.It seems reasonable to suggest that under oxidizing conditions (e.g.oxidative stress by ROS) formation of NOS/SONOS bridges in proteins, where the lysine and cysteine residues have direct roles in catalysis, goes along with a (reversible) loss of function.
In case of direct roles in the binding of enzymatic substrates, effectors or DNA, NOS bridge formation would lead to a reduced affinity and with that a diminished biological activity, although the extent may vary depending on the structural context.
For allosteric redox switches, both loss-of-function as well as gain-of-function scenarios seem possible with a variable modulation of biological function.In support of this proposal, the available biochemical data for key redox-sensitive proteins with identified NOS/SONOS redox switches indeed indicate a loss of function for proteins with catalytic lysines or cysteines as in case of e.g.DNA repair enzyme OGG1 (Schiff-base lysine) 22 or ubiquitin E2 ligases (catalytic cysteine) 23 under oxidative stress conditions (reaction with H2O2).For numerous other cases, it has been established that either reducing conditions are required for full biological activity, or that oxidizing conditions deteriorate protein activity 9,[24][25][26] .In case of galectin-1, which possesses an allosteric SONOS switch, it was established that the protein exerts different biological functions under oxidizing and reducing conditions and that binding of effectors requires the reduced state 27,28 .For the human protein, the two corresponding cysteines engaged in forming the SONOS bridge in the R. norvegicus ortholog (Fig. 2h) were identified as the redox switch site and suggested to form a disulfide although the latter could not be directly demonstrated 28 .The potential role of NOS redox switches in regulating DNA binding might provide a novel mechanism for the well established redox-dependent action of many transcription factors 8,29 .A putative regulatory role of the detected NOS/SONOS redox switches is further supported by the high degree of conservation of the lysine-cysteine pairs in the protein orthologues (either across different domains of life or in clades of related organisms) despite the fact that in many proteins only one of the two residues has a direct functional role required for biological activity as reported for e.g.OGG1 or rabphilin (SI Tables 3, 4) 30,31 .

NOS/SONOS bridges in proteins from pathogens and humans
We further classified the proteins with NOS/SONOS switches regarding their biological function.First, we analysed proteins from human and plant pathogens including viruses, bacteria and parasites (Fig. 7, ED Table 1).In case of pathogenic human viruses, NOS switch-containing proteins with essential roles for all critical phases of the virus life cycle such as infection (adenovirus), replication (SARS-CoV-2) and maturation assembly (cytomegalovirus) were detected.In bacteria and parasites, we identified numerous essential biosynthetic enzymes (amino acid,      ).This enzyme contains a serine (Ser332) at the equivalent position of Cys440 from M. tuberculosis DAH7P synthase and can therefore not form an NOS bridge.In the absence of the NOS bridge, the lysine forms a H-bond with the carboxylate moiety of substrate phosphoenolpyruvate.The structural models of key residues and substrate are superposed with the corresponding 2mFo-DFc electron density maps at 1σ. (f) Lysines with roles in binding of effectors.Left: Rabphilin 3a from Rattus norvegicus in complex with effector inositol trisphosphate (IP3) and with an NOS bridge formed between Lys423 and Cys473 (pdb code 4NP9).Ligand IP3 is weakly occupied with traceable density for the phosphate portions only.Lys423 is directly located at the binding site of IP3.The proximal Ca 2+ binding site is indicated.Right: Calexcitin from Loligo pealeii with an NOS bridge formed between Lys41 and Cys24 proximal to the Ca 2+ binding site (pdb code 2CCM).The structural models of the NOS bridge-forming lysine and cysteine residues are superposed with the corresponding 2mFo-DFc electron density maps at 1σ.    1 & 2.
Thus obtained steady-state activities were analyzed using the Michaelis-Menten equation and Hill equation.

Crystallisation, X-ray data collection, processing and model building
NgTAL wild-type and variant Glu93Gln were crystallized and cryoprotected as detailed in 9 .
Diffraction data of single protein crystals were collected using synchrotron radiation at beamline P14 of DESY EMBL, Hamburg, Germany, at a wavelength of either 0.9763 Å (NgTAL wild-type, dose series) or 0.689 Å (NgTAL variant Glu93Gln) at 100 K using an EIGER 16M detector.For crystals of NgTAL wild-type, we conducted a series of dosedependent dataset depositing calibrated doses (0.5-10 MGy in 0.5 MGy increments) per dataset.For data processing the XDS package was used 40 .Subsequent refinement and model building was performed employing PHENIX.REFINE 41 and COOT 42 .Phasing was performed by rigid body refinement using our previously determined NgTAL structure (pdb code 6ZX4) as initial model.The geometry of the structure was validated using MolProbity

43
. Representations of structures were prepared using PyMOL 44 .The Ramachandran statistics are 98.85% in the favoured and 1.15% in the allowed region for 7ODO, 98.85 % and 1.15 % for 7ODP, 98.85% in the favoured and 1.15% in the allowed region for 7ODQ and 98.55% in the favoured and 1.45% in the allowed region for 7OEY.

Computational Methods
We have carried out a series of electronic structure calculations to sample the conformational space and the relative energy of a model system consisting of a single lysine and cysteine residues.The residues were truncated at the α-carbon, with the latter represented as a methyl group to reduce the influence of electrostatics in the terminal moieties, closer to what is to expect from a protein backbone.Three different bonding situations were sampled.The first corresponds to a neutral lysine interacting with a cysteine (NHS).The second system considered was a protonated lysine with a cysteine (NHS+).Both cases will build close contacts through hydrogen bonds, either with the lysine as donor (NHS+) or the cysteine (NHS).In the last system we consider the covalently bound residues, with the lysine nitrogen and the cysteine sulfur bonding with an intercalated oxygen atom (NOS).
In a first set of calculations, we studied the dependence of the N-S-distances on the dielectric constant.To this purpose, a representative conformer for each of the systems was optimised in different solvents (vacuum: ε = 0.0, diehtylether: ε = 4.2, acetone: ε = 20.5, methanol: ε = 32.6,1,2-ethanediol:  = 40.2,dimethylsulfoxide:  = 46.8,water:  = 78.4) 45using the SMD 46 solvation model.The software package Gaussian16-A.03 47was used for the aforementioned calculations, with the B3LYP-D3(BJ)/def2-SVP 48-51 level of theory.A constrained α-carbon distance of 10 Å was applied for all the calculations with different solvents.The results are provided in SI Fig. 1.The only difference observed was a smaller difference between the distances observed in the NHS and NHS+ systems.Considering the stable behavior of the N-S-distances in different environments the following calculations were carried out in vacuum for simplicity and for a more straightforward generalisation.
Three different α-carbon distances (6, 8 and 10˚A) between lysine and cysteine were applied for the extended sampling runs (the results are provided in ED Figure 1).Initial conformer sampling was carried out using CREST-2.10.2 52 , whereby the conformer optimization was conducted with xTB-6.4.0 53 using the semiempirical tight-binding based quantum chemistry method GFN2-xTB 54 with the "extreme" optimization criteria (Econv = 5 × 10 −8 Hartree) and the "NCI" option.
After initial global sampling at the semiempirical level, the structures were refined in two stages at the density functional theory (DFT) level.For NOS, the collected GFN2-xTB conformer structures were further optimized using B3LYP-D3(BJ)/def2-SVP [48][49][50][51] , again with the use of the Gaussian16-A.03program package.Conformers with imaginary frequencies were directly abandoned and not used further for optimization or data statistics.For neutral and protonated hydrogen bonded systems (NHS and NHS+) the conformers were additionally filtered to guarantee the existence of hydrogen bonds and, thereby, afford the closest contacts possible.As criteria a distance shorter than 2.5 Å between the hydrogen (H) and the hydrogen acceptor (A) and an angle ADH (D: hydrogen donor) smaller than 30° were taken.Determination of the hydrogen atom for the building of the hydrogen bond follows the shortest distance among the potential hydrogen bonds between the amine and thiol residues.The conformations obtained from B3LYP-D3(BJ)/def2-SVP were in a further stage sorted according to their electronic energies from low to high.If the energy difference between two conformations was larger than 0.1 kcal/mol and the root-mean-square difference (RMSD) for the superimposed structures larger than 0.125 Å 55, the two conformations were considered to be unique and subject to further calculations.From this pool of structures, we considered the lowest-lying conformers (up to 2 kcal/mol in difference to the global minimum) and carried out further optimizations at the B3LYP-D3(BJ)/def2-TZVPP level of theory.For both NHS and NHD+ systems the additional filter process to verify the existence of hydrogen bonds was again applied.The results of the first (B3LYP-D3(BJ)/def2-SVP) and the second stage (B3LYP-D3(BJ)/def2-TZVPP) of optimizations were used and displayed in ED Figure 1.An overview of different geometric values and their correlation for the NOS bond is provided in SI Fig. 2.
In a further set of calculations, we investigated what could be the lowest energy conformer for both NHS and NHS+ bearing a N-S distance of 2.7 Å. By comparing the results of this sampling, we were able to assess the energy penalty associated with bringing the two residues in such a close vicinity without any covalent bond formation.This involved two further sampling rounds with the α-carbon distances set to 8 Å.The latter choice was considered as non-critical, given that the relative energetic and geometry information for the different bonding situations did not change dramatically upon variation of said parameter.Each sampling run was performed following the same steps as before, but with an added constraint to the N-S-distance of 2.7 Å.No additional filter process for hydrogen bonds was applied.RMSD criteria were also not applied since we were not interested in a distribution, but simply the most energetically stable geometry.The energies of the lowest conformers ("global" minima) were compared for the runs with and without the distance constraint.This resulted in an energy gap of 4.3 kcal/mol (SI Fig. 3).The thermochemical calculations for formation of the SONOS bridge are exemplified shown for the SARS-CoV-2-main protease Mpro (SI Fig. 4), which showcase a putative pathway for two subsequent oxidation steps.

Search of the Protein Data Bank for potential NOS or SONOS bridges
On December 2 nd 2020, we searched all deposites structures at the pdb (www.rcsb.org)with a resolution of 2 Å or better (65,327 out of ~170,000 structures) for potential NOS or SONOS bridges, using the program NCONT of the CCP4 suite 56 .In view of the quantum chemically calculated N-S interatomic distance of 2.6-2.7 Å for NOS bridges, we defined thresholds of 3.00 Å as upper limit and 2.45 Å as lower limit for the search.As non-covalent hydrogen-bond interactions between lysines and cysteines exhibit interatomic distances of >3.2 Å, we considered a cut-off at 3 Å to be a robust discriminator between covalent and non-covalent Cys-Lys interactions.A total of 285 pdb entries with ~400 potential NOS/SONOS bridges were identified, for 266 of these the corresponding structure factors were deposited.We examined all entries manually and inspected the potential NOS/SONOS redox switch sites.In case the electron density maps indicated the presence a covalent crosslink between the lysine N atom and the cysteine S atom, we rebuilt the structural model with an NOS/SONOS bridge and compared the obtained models and electron density maps with those calculated for structures with a non-covalent interaction between the lysine and cysteine side chains.Model building was carried out using COOT 42 , and refinements were done with PHENIX.REFINE 41 .

Analysis of sequence conservation
The analysis of sequence conservation of lysine and cysteine residues forming NOS or SONOS bridges was performed for the 1,000 closest related protein sequences obtained using BLASTP 57 for the respective protein of interest.Alignment was performed using MAFFT 58 .
isoprenoid and polyamine biosynthesis), Na + transporters required for ATP biosynthesis, transcription regulators, virulence factors and proteins in the context of pathogen-host interactions.Remarkably, numerous proteins originate from the world's most dangerous pathogens including i.a.SARS-CoV-2 (Covid 19), Pseudomonas aerigunosa (pneumonia), Staphylooccus aureus (bacterial superinfections), Legionella pneumophila (Legionnaires' disease) or Trypanosoma species (Chagas disease, African sleeping sickness).NOS bridges were also discovered in major plant pathogens including Xanthomonas campestris and Xanthomonas axonopodis, the causative agents of "black rot" in cruciferous vegetables and bacterial pustule of soybean32 .In humans, NOS redox switches are found in different protein families with established roles in oxidative stress response, redox signaling and homeostasis (Fig.7, ED Table2).Major families comprise proteins in DNA repair (excision of oxidized bases, cleavage of protein-DNA crosslinks), in transcription regulation (sensing and binding of DNA, histone writers and erasers), in ribosomal protein translation (translation factors), in protein degradation (ubiquitin E2 ligases, E3 ring ligase), in signaling (various kinases, neurotransmission, inositolphosphate signaling), in the biosynthesis of redox-sensitive cofactors and rare amino acids (Fe-S cluster, S-adenosyl-methionine, selenocysteine) (examples in Fig.6, ED Fig.3,5).Also, the enzyme selenophosphate synthetase 1 was identified which is known to be an essential factor of cellular redox homeostasis by regulating the expression levels of e.g.glutaredoxin and glutathione transferase, which protect cells from oxidative stress 33 .Many of the identified human proteins have been inferred in severe diseases including various cancers, Alzheimer's disease, Parkinson's disease, obesity, autoimmune diseases and others (ED Table2).The discovery of the naturally evolved NOS redox switches may therefore unlock novel therapeutic directions in plenty of disease states by addressing or manipulating the switch site.Prominent examples are e.g. the main protease from SARS-CoV-2 17,18 or human focal adhesion kinase, which are validated drug targets for treatment of COVID-19 and invasive cancers 34 , respectively.ConclusionsNOS and SONOS redox switches are ubiquitous regulatory elements in proteins that reversibly alter protein function in response to changing redox conditions as e.g.under oxidative stress.They are spontaneously formed without the need of catalysis provided by enzymes or proximal amino acids.The involvement of lysines with direct roles in enzyme catalysis and in binding of enzymatic substrates, nucleic acids and effectors expands the chemical repertoire of organisms to deal with changing redox conditions and constitutes a novel general regulatory principle in biology.Apart from novel directions in medical applications, the identified design principles of naturally evolved NOS/SONOS switches are likely to inspire peptide and protein design 35 , in particular the newly found branching SONOS crosslink bears great potential.

Figure 1 .
Figure 1.Chemical structures and topologies of NOS and SONOS redox switches in proteins.(a) Structures and suggested mechanism of NOS and SONOS redox bridge formation by ROS or O2 in subsequent oxidation steps.(b) Suggested structure of a 'mixed' NOS-disulfide redox switch, in which a disulfide is in equilibrium with an NOS bridge.(c) Topologies of NOS bridges showing intramolecular and intermolecular crosslinks as observed in experimentally determined protein structures.(d) Topologies of SONOS bridges showing intramolecular and intermolecular crosslinks as observed in experimentally determined protein structures.A SONOS bridge, where the lysine and two cysteines are contributed by three different proteins has not been identified yet.

Figure 2 .
Figure 2. Structural and chemical motifs of NOS bridges in proteins.(a) Histogram showing the distribution of detected proteins (non-redundant counts) with NOS bridges in dependence from the distance in sequence between the lysine and cysteine residues.(b) Examples for NOS bridges in intra-strand or strand-like motifs with a sequence distance of n+2.Shown are focal adhesion kinase from Gallus gallus (pdb code 6CBO) and ribose

Figure 3 .
Figure 3. NOS and SONOS bridges in the main protease Mpro from SARS-CoV-2.(a) Structure of Mpro in the reduced state (pdb code 7JR3) showing the redox switch at the protein surface formed by residues Cys22, Cys44 and Lys61 (highlighted in red) and the active site with residues Cys145 (catalytic nucleophile), His41 and Tyr54 (highlighted in slate blue).A mobile loop bearing Cys44 is indicated in magenta.Left panel: structural overview.Right panel: close-up of the redox switch site.For residues Cys22, Cys44 and Lys61, the structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ.Note that residue Cys44 is in the "in-conformation" and interacts with Tyr54.(b) Structure of Mpro in the mono-oxidized state with an NOS bridge formed between Cys22 and Lys61 (pdb code 6XMK).Left panel: structural overview.Right panel: close-up of the redox switch site.For residues Cys22, Cys44 and Lys61, the structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ.Cys44 is found in the "in-conformation".(b) Structure of Mpro in the di-oxidized state with a SONOS bridge formed between Cys22, Lys61 and Cys44 (pdb code 7JR4).Left panel: structural overview.Right panel: close-up of the redox switch site.For residues Cys22, Cys44 and Lys61, the structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ.Cys44 is found in the "out-conformation".Competitive refinements (SONOS bridge only, 2 separate NOS bridges, mixture of SONOS and NOS) indicate full occupancy of the SONOS bridge.

Figure 4 .
Figure 4. Functional roles of NOS bridge lysines in enzyme catalysis and in binding of enzymatic substrates or effectors.For (a-e), the left panels show the proteins in the oxidized state with the NOS bridge present, while the corresponding right panel shows the same or a closely related protein in the reduced state with the lysine exerting its function.(a) Catalytic

Figure 5 .
Figure5.Chemical functions of lysine and cysteine residues forming NOS and SONOS bridges in proteins.Four major functional categories could be identified including i) lysines with catalytic roles in enzyme mechanisms, ii) lysines involved in binding of enzyme substrates, nucleic acids and effectors, iii) cysteines with catalytic roles in enzyme mechanisms and iv) allosteric bridges, which are located remotely relative to the active/functional site.Structures of key reaction intermediates and interaction partners are highlighted.Note that these functions are exerted under reducing conditions that is in the absence of NOS/SONOS bridges.Formation of the NOS or SONOS bridge under oxidizing conditions leads to either a loss-of-function (catalytic lysines, catalytic cysteines), diminished biologial activity (lysine with binding roles) or modulated function (allosteric switches).

Figure 6 .
Figure 6.Proteins with NOS bridges inferred in DNA binding and transcription regulation.(a) DNA polymerase from Bacillus virus phi29 complexed with single-stranded DNA (pdb code 2PY5).Upper panel: Overall structure showing the two copies (chains a and b) in the asymmetric unit.The single stranded DNA is highlighted in salmon red.Lower panel: Closeup of the NOS redox switch with residues Lys114 and Cys106.Note that in chain A both residues form an NOS bridge (left), which is absent in chain B (right).In the absence of the NOS bridge, Lys 114 forms a H-bond with a DNA base via a water molecule.The structural models of the NOS bridge-forming lysine and cysteine residues are superposed with the

Figure 7 .
Figure 7. Occurrence and biological functions of proteins containing NOS and SONOS redox switches.Functions of proteins originating from human and plant pathogen are highlighted in blue color, representative examples of relevant species are shown alongside.Cellular functions of human proteins are highlighted in red color, functional subclasses and representative protein families are listed below.Specific information about all proteins regarding origin, biological function, type of NOS/SONOS redox switch, suggested mechanism of the redox switch and potential relevance in disease states is compiled in ED Tables 1 & 2.
lysines forming Schiff-base intermediates in PLP dependent enzymes.Left: Arginine decarboxylase from Paramecium bursaria Chlorella Virus (pdb code 2NV9), in which the catalytic Lys48 forms an NOS bridge with Cys324.Right: Ornithine decarboxylase from Trypanosoma brucei in covalent complex with product putrescine (pdb code 1F3T).The structural models are superposed with the corresponding 2mFo-DFc electron density maps at 1σ.Note that in the presence of the NOS bridge, the reaction does not proceed beyond the carbinolamine.(b) Catalytic lysines forming Schiff-base intermediates with enzymatic substrates.Left: KDPG aldolase from Oleispira antarctica in non-covalent complex with substrate pyruvate and with an NOS bridge between Lys136 and Cys162 (pdb code 3VCR).Right: KDPG aldolase from E. coli in covalent complex with substrate pyruvate (pdb code 1EUA).The structural models are superposed with the corresponding 2mFo-DFc electron density maps at 1σ.Note that in the presence of the NOS bridge, covalent catalysis is inhibited.(c) Catalytic lysines in carboxyl transfer.Left: Oxaloacetate decarboxylase/Na + pump from Vibrio cholerae with an NOS bridge between Lys178 and Cys148 (pdb code 2NX9).Right: Transcarboxylase 5S subunit from Propionibacterium freudenreichii with carboxylated Lys184 (pdb code 1RQB) thought to be an intermediate in CO2 transfer to biotin.The structural models are superposed with the corresponding 2mFo-DFc electron density maps at 1σ.Note that in the presence of the NOS bridge, catalysis is inhibited.(d) Catalytic lysines acting as acid-base catalysts.Left: Penicillin binding protein (transpeptidase) from Streptomyces sp.K15 with an NOS bridge between Lys38 and Cys98 (pdb code 1SKF).Right: Penicillin binding protein variant Cys98Ala from Streptomyces sp.K15 (pdb code 1ES3).As Lys38 is thought to activate the nucleopilic Ser35 by acid-base catalysis, formation of an NOS bridge under oxidizing conditions is likely to inhibit catalysis.The structural models of residues 38 and 98 are superposed with the corresponding 2mFo-DFc electron density maps at 1σ. (e) Lysines with roles in non-covalent binding of enzymatic substrates.Left: DAH7P synthase from Mycobacterium tuberculosis (pdb code 3RZI) with an NOS bridge between Lys133 and Cys440.Right: DAH7P synthase from Listeria monocytogenes in non-covalent complex with substrate phosphoenolpyruvate (pdb code 3TFC