Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction.

Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the ß-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/ß dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.

Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Genetically Encoded Biosensors for the Fluorescence Detection of O2 and Reactive O2 Species.

The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies.

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics.

Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.

Rational Design of a User-Friendly Aptamer/Peptide-Based Device for the Detection of Staphylococcus aureus.

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.

Iron Metabolism at the Interface between Host and Pathogen: From Nutritional Immunity to Antibacterial Development.

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery.

Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States.

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase-the enzyme responsible for allantoin hydrolization in plants and lower organisms-for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein-protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form.

Serine racemase is the pyridoxal 5'-phosphate dependent enzyme that catalyzes both production and catabolism of d-serine, a co-agonist of the NMDA glutamate receptors. Mg2+, or, alternatively, Ca2+, activate human serine racemase by binding both at a specific site and - as ATP-metal complexes - at a distinct ATP binding site. We show that Mg2+ and Ca2+ bind at the metal binding site with a 4.5-fold difference in affinity, producing a similar thermal stabilization and partially shifting the dimer-tetramer equilibrium in favour of the latter. The ATP-Ca2+ complex produces a 2-fold lower maximal activation in comparison to the ATP-Mg2+ complex and exhibits a 3-fold higher EC50. The co-presence of ATP and metals further stabilizes the tetramer. In consideration of the cellular concentrations of Mg2+ and Ca2+, even taking into account the fluctuations of the latter, these results point to Mg2+ as the sole physiologically relevant ligand both at the metal binding site and at the ATP binding site. The stabilization of the tetramer by both metals and ATP-metal complexes suggests a quaternary activation mechanism mediated by 5'-phosphonucleotides similar to that observed in the distantly related prokaryotic threonine deaminases. This allosteric mechanism has never been observed before in mammalian fold type II pyridoxal 5'-phosphate dependent enzymes.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

PURPOSE: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the ß-subunit (F41). We therefore replaced this residue with a tyrosine (ßF41Y, Hb Mequon). The ßF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, ßF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the ß-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in ßF41Y. NO bioavailability was enhanced in ßF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the ß-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.

Experimental basis for a new allosteric model for multisubunit proteins.

Monod, Wyman, and Changeux (MWC) explained allostery in multisubunit proteins with a widely applied theoretical model in which binding of small molecules, so-called allosteric effectors, affects reactivity by altering the equilibrium between more reactive (R) and less reactive (T) quaternary structures. In their model, each quaternary structure has a single reactivity. Here, we use silica gels to trap protein conformations and a new kind of laser photolysis experiment to show that hemoglobin, the paradigm of allostery, exhibits two ligand binding phases with the same fast and slow rates in both R and T quaternary structures. Allosteric effectors change the fraction of each phase but not the rates. These surprising results are readily explained by the simplest possible extension of the MWC model to include a preequilibrium between two tertiary conformations that have the same functional properties within each quaternary structure. They also have important implications for the long-standing question of a structural explanation for the difference in hemoglobin oxygen affinity of the two quaternary structures.

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models.

Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include pre-equilibria of tertiary as well as quaternary conformations; the structure-based model of Szabo and Karplus; and the modification of the latter model by Lee and Karplus. We show that only the tertiary two-state model can provide a near quantitative explanation of the single-crystal and gel experimental results.

Molecular insights into dimerization inhibition of c-Maf transcription factor.

The Maf protein family belongs to the activator protein 1 (AP-1) superfamily of transcription factors that bind specific DNA target sequences through a basic region and exploit a leucine zipper (LZ) motif for protein-protein interactions leading to homo- or hetero-dimerization. Mafs unique DNA-binding domain contains a highly conserved extended homology region (EHR) that allows to recognize longer DNA sequences than other basic leucine zipper (bZIP) transcription factors. Inspired by the fact that overexpression of Mafs is observed in about 50% of cases of multiple myeloma, a hematological malignant disorder, we undertook a peptide inhibitor approach. The LZ domain of c-Maf, one of large Mafs, was produced by solid phase peptide synthesis. We characterized its secondary structure and dimerization properties, and found that dimerization and folding events are strictly coupled. Moreover, potential peptidic c-Maf dimerization inhibitors were computationally designed and synthesized. These compounds were demonstrated by circular dichroism (CD) spectroscopy and MALDI-TOF mass spectrometry to bind to c-Maf LZ monomers, to drive folding of their partially disordered structure and to efficiently compete with dimerization, suggesting a way for interfering with the function of c-Maf and, more generally, of intrinsically disordered proteins, till now considered undruggable targets.

MediaChrom: Discovering a Class of Pyrimidoindolone-Based Polarity-Sensitive Dyes.

A small library of six polarity-sensitive fluorescent dyes, nicknamed MediaChrom, was prepared. This class of dyes is characterized by a pyrimidoindolone core fitted out with a conjugated push-pull system and a carboxy linker for a conceivable coupling with biomolecules. The optimized eight-step synthetic strategy involves a highly chemo- and regioselective gold-catalyzed cycloisomerization reaction. The photophysical properties of MediaChrom dyes have been evaluated in-depth. In particular, the MediaChrom bearing a diethylamino as an electron-donating group and a trifluoromethyl as an electron-withdrawing group displays the most interesting and advantageous spectroscopic features (e.g., absorption and emission in the visible range and a good quantum yield). Promising results in terms of sensitivity have been obtained in vitro on this dye as a membrane/lipophilic probe and as a peptide fluorescent label.

Tertiary and quaternary effects in the allosteric regulation of animal hemoglobins.

In the last decade, protein allostery has experienced a major resurgence, boosted by the extension of the concept to systems of increasing complexity and by its exploitation for the development of drugs. Expansion of the field into new directions has not diminished the key role of hemoglobin as a test molecule for theory and experimental validation of allosteric models. Indeed, the diffusion of hemoglobins in all kingdoms of life and the variety of functions and of quaternary assemblies based on a common tertiary fold indicate that this superfamily of proteins is ideally suited for investigating the physical and molecular basis of allostery and firmly maintains its role as a main player in the field. This review is an attempt to briefly recollect common and different strategies adopted by metazoan hemoglobins, from monomeric molecules to giant complexes, exploiting homotropic and heterotropic allostery to increase their functional dynamic range. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Role of tertiary structures on the Root effect in fish hemoglobins.

Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH6.2 and pH8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T-R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.