Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

Best Practices on QM/MM Simulations of Biological Systems.

During the second half of the 20th century, following structural biology hallmark works on DNA and proteins, biochemists shifted their questions from "what does this molecule look like?" to "how does this process work?". Prompted by the theoretical and practical developments in computational chemistry, this led to the emergence of biomolecular simulations and, along with the 2013 Nobel Prize in Chemistry, to the development of hybrid QM/MM methods. QM/MM methods are necessary whenever the problem we want to address involves chemical reactivity and/or a change in the system's electronic structure, with archetypal examples being the studies of an enzyme's reaction mechanism and a metalloprotein's active site. In the last decades QM/MM methods have seen an increasing adoption driven by their incorporation in widely used biomolecular simulation software. However, properly setting up a QM/MM simulation is not an easy task, and several issues need to be properly addressed to obtain meaningful results. In the present work, we describe both the theoretical concepts and practical issues that need to be considered when performing QM/MM simulations. We start with a brief historical perspective on the development of these methods and describe when and why QM/MM methods are mandatory. Then we show how to properly select and analyze the performance of the QM level of theory, the QM system size, and the position and type of the boundaries. We show the relevance of performing prior QM model system (or QM cluster) calculations in a vacuum and how to use the corresponding results to adequately calibrate those derived from QM/MM. We also discuss how to prepare the starting structure and how to select an adequate simulation strategy, including those based on geometry optimizations as well as free energy methods. In particular, we focus on the determination of free energy profiles using multiple steered molecular dynamics (MSMD) combined with Jarzynski's equation. Finally, we describe the results for two illustrative and complementary examples: the reaction performed by chorismate mutase and the study of ligand binding to hemoglobins. Overall, we provide many practical recommendations (or shortcuts) together with important conceptualizations that we hope will encourage more and more researchers to incorporate QM/MM studies into their research projects.

Type 1 Insulin-Like Growth Factor Receptor Nuclear Localization in High-Grade Glioma Cells Enhances Motility, Metabolism, and In Vivo Tumorigenesis.

Gliomas are the most frequent solid tumors in children. Among these, high-grade gliomas are less common in children than in adults, though they are similar in their aggressive clinical behavior. In adults, glioblastoma is the most lethal tumor of the central nervous system. Insulin-like growth factor 1 receptor (IGF1R) plays an important role in cancer biology, and its nuclear localization has been described as an adverse prognostic factor in different tumors. Previously, we have demonstrated that, in pediatric gliomas, IGF1R nuclear localization is significantly associated with high-grade tumors, worst clinical outcome, and increased risk of death. Herein we explore the role of IGF1R intracellular localization by comparing two glioblastoma cell lines that differ only in their IGF1R capacity to translocate to the nucleus. In vitro, IGF1R nuclear localization enhances glioblastoma cell motility and metabolism without affecting their proliferation. In vivo, IGF1R has the capacity to translocate to the nucleus and allows not only a higher proliferation rate and the earlier development of tumors but also renders the cells sensitive to OSI906 therapy. With this work, we provide evidence supporting the implications of the presence of IGF1R in the nucleus of glioma cells and a potential therapeutic opportunity for patients harboring gliomas with IGF1R nuclear localization.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Specificity and Reactivity of Mycobacterium tuberculosis Serine/Threonine Kinases PknG and PknB.

Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine protein kinases, which play essential roles in cell growth, signal transduction, and pathogenesis. Protein kinase G (PknG) regulates the carbon and nitrogen metabolism by phosphorylation of the glycogen accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB) is involved in cell wall synthesis and cell shape, as well as phosphorylates GarA but at Thr22. While PknG seems to be constitutively activated and recognition of GarA requires phosphorylation in its unstructured tail, PknB activation is triggered by phosphorylation of its activation loop, which allows binding of the forkhead-associated domain of GarA. In the present work, we used molecular dynamics and quantum-mechanics/molecular mechanics simulations of the catalytically competent complex and kinase activity assays to understand PknG/PknB specificity and reactivity toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem essential for PknG binding and allow specificity for Thr21 phosphorylation. On the other hand, phosphorylated residues in PknB bind Arg26 in GarA and regulate its specificity for Thr22. We also provide a detailed analysis of the free energy profile for the phospho-transfer reaction and show why PknG has a constitutively active conformation not requiring priming phosphorylation in contrast to PknB. Our results provide new insights into these two key enzymes relevant for Mtb and the mechanisms of serine/threonine phosphorylation in bacteria.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.

Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO2.

Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO2. Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and ß-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-affinities, there is considerable variation in sensitivity to Cl- ions and ATP, which appears to be at least partly attributable to variation in the extent of NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.

Multiscale approach to the activation and phosphotransfer mechanism of CpxA histidine kinase reveals a tight coupling between conformational and chemical steps.

Sensor histidine kinases (SHKs) are an integral component of the molecular machinery that permits bacteria to adapt to widely changing environmental conditions. CpxA, an extensively studied SHK, is a multidomain homodimeric protein with each subunit consisting of a periplasmic sensor domain, a transmembrane domain, a signal-transducing HAMP domain, a dimerization and histidine phospho-acceptor sub-domain (DHp) and a catalytic and ATP-binding subdomain (CA). The key activation event involves the rearrangement of the HAMP-DHp helical core and translation of the CA towards the acceptor histidine, which presumably results in an autokinase-competent complex. In the present work we integrate coarse-grained, all-atom, and hybrid QM-MM computer simulations to probe the large-scale conformational reorganization that takes place from the inactive to the autokinase-competent state (conformational step), and evaluate its relation to the autokinase reaction itself (chemical step). Our results highlight a tight coupling between conformational and chemical steps, underscoring the advantage of CA walking along the DHp core, to favor a reactive tautomeric state of the phospho-acceptor histidine. The results not only represent an example of multiscale modelling, but also show how protein dynamics can promote catalysis.

Structural and mechanistic comparison of the Cyclopropane Mycolic Acid Synthases (CMAS) protein family of Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic disease caused by the bacillus Mycobacterium tuberculosis(Mtb) and remains a leading cause of mortality worldwide. The bacteria has an external wall which protects it from being killed, and the enzymes involved in the biosynthesis of the cell wall components have been proposed as promising targets for future drug development efforts. Cyclopropane Mycolic Acid Synthases (CMAS) constitute a group of ten homologous enzymes which belong to the mycolic acid biosynthesis pathway. These enzymes have S-adenosyl-l-methionine (SAM) dependent methyltransferase activity with a peculiarity, each one of them has strong substrate selectivity and reaction specificity, being able to produce among other things cyclopropanes or methyl-alcohol groups from the lipid olefin group. How each CMAS processes its substrate and how the specificity and selectivity are encoded in the protein sequence and structure, is still unclear. In this work, by using a combination of modeling tools, including comparative modeling, docking, all-atom MD and QM/MM methodologies we studied in detail the reaction mechanism of cmaA2, mmaA4, and mmaA1 CMAS and described the molecular determinants that lead to different products. We have modeled the protein-substrate complex structure and determined the free energy pathway for the reaction. The combination of modeling tools at different levels of complexity allows having a complete picture of the CMAS structure-activity relationship.

Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8+ cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.

Theoretical Insights into the Reaction and Inhibition Mechanism of Metal-Independent Retaining Glycosyltransferase Responsible for Mycothiol Biosynthesis.

Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus, the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center, and therefore, they are classified as either inverting or retaining GTs. While the inverting GT reaction mechanism has been widely studied, the retaining GT mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNAN in IUPAC nomenclature) and has a 17.5 kcal/mol free energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion-like transition state. In contrast, fluorinated substrate analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the transition state.

Heme oxygenase-1 in the forefront of a multi-molecular network that governs cell-cell contacts and filopodia-induced zippering in prostate cancer.

Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

Discussing endogenous NO(•)/HNO interconversion aided by phenolic drugs and vitamins.

The reduction of NO(•) to HNO/NO(-) under biologically compatible conditions has always been thought as unlikely, mostly because of the negative reduction potential: E°(NO(•),H(+)/HNO) = -0.55 V vs NHE at physiological pH. Nonetheless, during the past decade, several works hinted at the possible NO-to-HNO conversion mediated by moderate biological reductants. Very recently, we have shown that the reaction of NO(•) with ascorbate and aromatic alcohols occurs through a proton-coupled nucleophilic attack (PCNA) of the alcohol to NO(•), yielding an intermediate RO-N(H)O(•) species, which further decomposes to release HNO. For the present work, we decided to inspect whether other common biological aromatic alcohols obtained from foods, such as Vitamin E, or used as over-the-counter drugs, like aspirin, are able to undergo the reaction. The positive results suggest that the conversion of NO to HNO could occur far more commonly than previously expected. Taking these as the starting point, we set to review our and other groups' previous reports on the possible NO-to-HNO conversion mediated by biological compounds including phenolic drugs and vitamins, as well as several thiol-bearing compounds. Analysis of revised data prompted us to ask ourselves the following key questions: What are the most likely physio/pathological conditions for NO(•)-to-HNO conversion to take place? Which effects usually attributed to NO(•) are indeed mediated by HNO? These inquiries are discussed in the context of 2 decades of NO and HNO research.

Protein topology determines cysteine oxidation fate: the case of sulfenyl amide formation among protein families.

Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.

Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.