A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1-4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch9,10.

Low-barrier hydrogen bonds in enzyme cooperativity.

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins, with haemoglobin and aspartate transcarbamoylase serving as prototypical examples1,2. The binding of effectors typically causes a structural transition of the protein that is propagated through signalling pathways to remote sites and involves marked changes on the tertiary and sometimes even the quaternary level1-5. However, the origin of these signals and the molecular mechanism of long-range signalling at an atomic level remain unclear5-8. The different spatial scales and timescales in signalling pathways render experimental observation challenging; in particular, the positions and movement of mobile protons cannot be visualized by current methods of structural analysis. Here we report the experimental observation of fluctuating low-barrier hydrogen bonds as switching elements in cooperativity pathways of multimeric enzymes. We have observed these low-barrier hydrogen bonds in ultra-high-resolution X-ray crystallographic structures of two multimeric enzymes, and have validated their assignment using computational calculations. Catalytic events at the active sites switch between low-barrier hydrogen bonds and ordinary hydrogen bonds in a circuit that consists of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons by behaving as an atomistic Newton's cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide several lines of evidence and a working model for not only the existence of low-barrier hydrogen bonds in proteins, but also a connection to enzyme cooperativity. This finding suggests new principles of drug and enzyme design, in which sequences of residues can be purposefully included to enable long-range communication and thus the regulation of engineered biomolecules.

Widespread occurrence of covalent lysine-cysteine redox switches in proteins.

We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous 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 sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. 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 as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.

Modulating Secondary Structure Motifs Through Photo-Labile Peptide Staples.

Peptide-protein interactions (PPIs) are facilitated by the well-defined three-dimensional structure of bioactive peptides, interesting compounds for the development of new therapeutic agents. Their secondary structure and thus their propensity to engage in PPIs can be influenced by the introduction of peptide staples on the side chains. In particular, light-controlled staples based on azobenzene photoswitches and their structural influence on helical peptides have been studied extensively. In contrast, photolabile staples bearing photocages as a structural key motif, have mainly been used to block supramolecular interactions. Their influence on the secondary structure of the target peptide is under-investigated. Thus, in this study we use a combination of spectroscopic techniques and in silico simulations to systematically study a series of helical peptides with varying length of the photo-labile staple to obtain a detailed insight into the structure-property relationship in such photoresponsive biomolecules.

One- and Two-Electron Reductions in MiniSOG and their Implication in Catalysis.

The unconventional bioorthogonal catalytic activation of anticancer metal complexes by flavin and flavoproteins photocatalysis has been reported recently. The reactivity is based on a two-electron redox reaction of the photoactivated flavin. Furthermore, when it comes to flavoproteins, we recently reported that site mutagenesis can modulate and improve this catalytic activity in the mini Singlet Oxygen Generator protein (SOG). In this paper, we analyze the reductive half-reaction in different miniSOG environments by means of density functional theory. We report that the redox properties of flavin and the resulting reactivity of miniSOG is modulated by specific mutations, which is in line with the experimental results in the literature. This modulation can be attributed to the fundamental physicochemical properties of the system, specifically (i) the competition of single and double reduction of the flavin and (ii) the probability of electron transfer from the protein to the flavin. These factors are ultimately linked to the stability of flavin's electron-accepting orbitals in different coordination modes.

The Catalytic Mechanism of Acetoacetate Decarboxylase: A Detailed Study of Schiff Base Formation, Protonation States, and Their Impact on Catalysis.

The enzyme acetoacetate decarboxylase (AAD) has a crucial function in the process of decarboxylating the substrate acetoacetate (AA). It has been extensively studied over the years, but its exact catalytic mechanism has remained partly unsolved due to the difficulty in assessing reaction intermediates. In this study, we combine molecular dynamics and electronic structure calculations to rediscover its catalytic mechanism. Our results show that the presence of the substrate, the acetoacetate, significantly influences the electrostatic potential of the active site. Furthermore, our simulations show that the decarboxylation reaction can take place by means of a direct proton transfer instead of via an enamine intermediate, which is thought to be strictly necessary. This work provides new insights into the role of the electrostatic interactions on the catalytic activity of AAD and for the first time connects it to the catalytic mechanism of other decarboxylases.

Mechanisms of Cysteine-Lysine Covalent Linkage-The Role of Reactive Oxygen Species and Competition with Disulfide Bonds.

Recently, a new naturally occurring covalent linkage was characterised, involving a cysteine and a lysine, bridged through an oxygen atom. The latter was dubbed as the NOS bond, reflecting the individual atoms involved in this uncommon bond which finds little parallel in lab chemistry. It is found to form under oxidising conditions and is reversible upon addition of reducing agents. Further studies have identified the bond in crystal structures across a variety of systems and organisms, potentially playing an important role in regulation, cellular defense and replication. Not only that, double NOS bonds have been identified and even found to be competitive in relation to the formation of disulfide bonds. This raises several questions about how this exotic bond comes to be, what are the intermediates involved in its formation and how it competes with other pathways of sulfide oxidation. With this objective in mind, we revisited our first proposed mechanism for the reaction with model electronic structure calculations, adding information about the reactivity with alternative reactive oxygen species and other potential competing products of oxidation. We present a network with more than 30 reactions which provides one of the most encompassing pictures for cysteine oxidation pathways to date.

Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome.

The 20S proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells and in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome (caspase-like), an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy- and boronic acid-containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics, and Bayesian optimization of nonbonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach for the reevaluation of nonbonded potentials making use of the hybrid quantum mechanics molecular mechanics dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the studied inhibitors.

Can System Truncation Speed up Ligand-Binding Calculations with Periodic Free-Energy Simulations?

We have investigated whether alchemical free-energy perturbation calculations of relative binding energies can be sped up by simulating a truncated protein. Previous studies with spherical nonperiodic systems showed that the number of simulated atoms could be reduced by a factor of 26 without affecting the calculated binding free energies by more than 0.5 kJ/mol on average ( Genheden, S.; Ryde, U. J. Chem. Theory Comput. 2012 , 8 , 1449 ), leading to a 63-fold decrease in the time consumption. However, such simulations are rather slow, owing to the need of a large cutoff radius for the nonbonded interactions. Periodic simulations with the electrostatics treated by Ewald summation are much faster. Therefore, we have investigated if a similar speed-up can be obtained also for periodic simulations. Unfortunately, our results show that it is harder to truncate periodic systems and that the truncation errors are larger for these systems. In particular, residues need to be removed from the calculations, which means that atoms have to be restrained to avoid that they move in an unrealistic manner. The results strongly depend on the strength on this restraint. For the binding of seven ligands to dihydrofolate reductase and ten inhibitors of blood-clotting factor Xa, the best results are obtained with a small restraining force constant. However, the truncation errors were still significant (e.g., 1.5-2.9 kJ/mol at a truncation radius of 10 Å). Moreover, the gain in computer time was only modest. On the other hand, if the snapshots are truncated after the MD simulations, the truncation errors are small (below 0.9 kJ/mol even for a truncation radius of 10 Å). This indicates that postprocessing with a more accurate energy function (e.g., with quantum chemistry) on truncated snapshots may be a viable approach.

Photosensitization mechanism of Cu(ii) porphyrins.

This work presents the mechanism of the photoinduced generation of reactive oxygen species (ROS) by paramagnetic copper porphyrins in aqueous solution. Electronic structure calculations within the framework of the (time-dependent) density functional theory, (TD)DFT, reveal the details regarding the development of the atomistic and electronic structures of the copper porphyrin in solution along the set of chemical reactions accessible upon photoactivation. This study identifies the key parameters controlling the feasibility of the various reaction pathways that drive the formation of specific reactive oxygen species, ROS, i.e. superoxide, peroxyl and hydroxyl radicals. An important outcome of our results is the rationalization of how the water solvent molecules play a crucial role in most steps of the overall reaction. The present study is illustrated by focusing on one specific copper porphyrin for which precise experimental data have recently been measured, and can readily be generalized to the whole family of paramagnetic porphyrins. The conclusions of this work shed light on the rational design of metalloporphyrins as photosensitizers for photodynamic therapy.

A computational study of radical initiated protein backbone homolytic dissociation on all natural amino acids.

Hydroxyl radical (˙OH) is known to be one of the most reactive species. In this work, the hydrogen abstraction by ˙OH from Cα and Cß atoms of all amino acids is studied in the framework of density functional theory as this is the most favorable reaction mechanism when this kind of radical attacks a protein. From the myriad routes that the oxidation of a protein by a ˙OH radical may follow, fragmentation of the protein is one of the most damaging ones as it hampers the normal function of the protein. Therefore, cleavages of the Cα-C and Cα-N backbone bonds have been investigated as the second step of the mechanism. To the best of our knowledge, this is the first time that this reaction pathway has been systematically studied for all natural amino acids. The study includes the effects that the solvent dielectrics or the conformation of the peptide model employed has on the reaction. Interestingly, the results indicate that the nature of the side chain has little effect on the H abstraction reaction, and that for most of amino acids the attack at the Cα atom is favored over the attack at the Cß atom. The origin of this preference relies on the larger capability of the formed radical intermediate to delocalize the unpaired electron, thus maximizing the captodative effect. Moreover, the reaction is more favorable when the reactant presents a ß-sheet conformation, with a completely planar peptide backbone. With respect to the homolytic splitting of the Cα-C and Cα-N bonds, the former is favorable for almost all amino acids, whereas Ser and Thr are the only amino acids favoring the latter. These results agree with previous investigations but an accurate description of the electronic density analysis performed indicates that the origin of the different reaction pathway preferences relies on a large stabilization of the product rather than bond weakening at the radical intermediate.

Multiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design.

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

Dynamic Protonation States Underlie Carbene Formation in ThDP-Dependent Enzymes: A Theoretical Study.

The activation mechanism of thiamine diphosphate (ThDP) in enzymes has long been the subject of intense research and controversial discussion. Particularly contentious is the formation of a carbene intermediate, the first one observed in an enzyme. For the formation of the carbene to take place, both intramolecular and intermolecular proton transfer pathways have been proposed. However, the physiologically relevant pH of ThDP-dependent enzymes around neutrality does not seem to be suitable for the formation of such reactive chemical species. Herein, we investigate the general mechanism of activation of the ThDP cofactor in human transketolase (TKT), by means of electronic structure methods. We show that in the case of the human TKT, the carbene species is accessible through a pKa shift induced by the electrostatics of a neighboring histidine residue (H110), whose protonation state change modulates the pKa of ThDP and suppresses the latter by more than 6 pH units. Our findings highlight that ThDP enzymes activate the cofactor beyond simple geometric constraints and the canonical glutamate. Such observations in nature can pave the way for the design of biomimetic carbene catalysts and the engineering of tailored enzymatic carbenes.

The nature of chemical bonds from PNOF5 calculations.

Natural orbital functional theory (NOFT) is used for the first time in the analysis of different types of chemical bonds. Concretely, the Piris natural orbital functional PNOF5 is used. It provides a localization scheme that yields an orbital picture which agrees very well with the empirical valence shell electron pair repulsion theory (VSEPR) and Bent's rule, as well as with other theoretical pictures provided by valence bond (VB) or linear combination of atomic orbitals-molecular orbital (LCAO-MO) methods. In this context, PNOF5 provides a novel tool for chemical bond analysis. In this work, PNOF5 is applied to selected molecules that have ionic, polar covalent, covalent, multiple (σ and π), 3c-2e, and 3c-4e bonds.

Oxidation of Acid, Base, and Amide Side-Chain Amino Acid Derivatives via Hydroxyl Radical.

Hydroxyl radical (•OH) is known to be highly reactive. Herein, we analyze the oxidation of acid (Asp and Glu), base (Arg and Lys), and amide (Asn and Gln) containing amino acid derivatives by the consecutive attack of two •OH. In this work, we study the reaction pathway by means of density functional theory. The oxidation mechanism is divided into two steps: (1) the first •OH can abstract a H atom or an electron, leading to a radical amino acid derivative, which is the intermediate of the reaction and (2) the second •OH can abstract another H atom or add itself to the formed radical, rendering the final oxidized products. The studied second attack of •OH is applicable to situations where high concentration of •OH is found, e.g., in vitro. Carbonyls are the best known oxidation products for these reactions. This work includes solvent dielectric and confirmation's effects of the reaction, showing that both are negligible. Overall, the most favored intermediates of the oxidation process at the side chain correspond to the secondary radicals stabilized by hyperconjugation. Intermediates show to be more stable in those cases where the spin density of the unpaired electron is lowered. Alcohols formed at the side chains are the most favored products, followed by the double-bond-containing ones. Interestingly, Arg and Lys side-chain scission leads to the most favored carbonyl-containing oxidation products, in line with experimental results.

Detecting the presence-absence of bluefin tuna by automated analysis of medium-range sonars on fishing vessels.

This study presents a methodology for the automated analysis of commercial medium-range sonar signals for detecting presence/absence of bluefin tuna (Tunnus thynnus) in the Bay of Biscay. The approach uses image processing techniques to analyze sonar screenshots. For each sonar image we extracted measurable regions and analyzed their characteristics. Scientific data was used to classify each region into a class ("tuna" or "no-tuna") and build a dataset to train and evaluate classification models by using supervised learning. The methodology performed well when validated with commercial sonar screenshots, and has the potential to automatically analyze high volumes of data at a low cost. This represents a first milestone towards the development of acoustic, fishery-independent indices of abundance for bluefin tuna in the Bay of Biscay. Future research lines and additional alternatives to inform stock assessments are also discussed.

·OH Oxidation Toward S- and OH-Containing Amino Acids.

The hydroxyl radical is the most reactive oxygen species, and it is able to attack macromolecules such as proteins. Such oxidation processes are the cause of a number of diseases. Several oxidized products have been experimentally characterized, but the reaction pathways remain unclear. Herein, we present a theoretical study on the attack of hydroxyl radicals on hydroxyl- and sulfur-containing amino acid side chains. Several reaction mechanisms, such as hydrogen abstraction, electron transfer, or ·OH addition have been considered to investigate several reaction mechanisms. Two different dielectric values (4 and 80) have been used to model the effect of different protein environments. In addition, different alternative conformations of the amino acid backbone have been considered. Overall, the results indicate that the thermodynamics is the main factor driving the reaction pathway preference and, to a great extent, explains the formation of the experimental oxidized produts. Sulfur-containing amino acids would be oxidized more easily than OH-containing amino acids, which confirms the experimental evidence. This is determined by the stability of the sulfur radical intermediates. These results are not dramatically affected by either different dielectrics or backbone conformations.