Is allostery a fuzzy concept?

Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two-state concerted model, but also those that obey different reaction mechanisms such as ligand-induced fit, possibly coupled to sequential structure changes, and ligand-linked dissociation-association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible 'allosteries'. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible.

Structural Basis of Sequential and Concerted Cooperativity.

Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the "concerted model" by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the "sequential model" originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms.

Hemoglobin allostery and pharmacology.

The oxygen demands of the human body require the constant circulation of blood carrying an enormous concentration of hemoglobin (Hb). Oxygen transport depends not only on the amount of Hb, but also on the control over the affinity of the protein for the gas, which can be optimized for the environmental conditions by changes in the concentration of effectors (hydrogen ions, chloride, CO2, and DPG) inside the red cell. Some pathological conditions affecting Hb may benefit from pharmacological interventions to increase or decrease its affinity for oxygen, or otherwise modify its properties, or alter its biosynthesis. Examples of such conditions include sickle cell anemia, thalassemias and inherited hemoglobinopathies. Effective and safe drugs such as voxelotor, bezafibrate and efaproxiral are available that significantly increase or decrease Hb oxygen affinity. Some medical conditions not directly affecting the blood or its oxygen carrying capacity may also be relieved by the manipulation of Hb. For example, the standard treatment of acute cyanide poisoning requires the oxidation of a fraction of the Hb in the bloodstream so that it efficiently scavenges cyanide. Tumors are often extremely hypoxic and therefore strongly resistant to radiotherapy; the sensitivity of cancerous tissue to X-rays may be increased by improved oxygenation through drugs binding Hb. This review attempts to provide a systematic exploration of the pharmacology of Hb, its molecular basis, and its intended and possible uses.

Mismatch between Tissue Partial Oxygen Pressure and Near-Infrared Spectroscopy Neuromonitoring of Tissue Respiration in Acute Brain Trauma: The Rationale for Implementing a Multimodal Monitoring Strategy.

The brain tissue partial oxygen pressure (PbtO2) and near-infrared spectroscopy (NIRS) neuromonitoring are frequently compared in the management of acute moderate and severe traumatic brain injury patients; however, the relationship between their respective output parameters flows from the complex pathogenesis of tissue respiration after brain trauma. NIRS neuromonitoring overcomes certain limitations related to the heterogeneity of the pathology across the brain that cannot be adequately addressed by local-sample invasive neuromonitoring (e.g., PbtO2 neuromonitoring, microdialysis), and it allows clinicians to assess parameters that cannot otherwise be scanned. The anatomical co-registration of an NIRS signal with axial imaging (e.g., computerized tomography scan) enhances the optical signal, which can be changed by the anatomy of the lesions and the significance of the radiological assessment. These arguments led us to conclude that rather than aiming to substitute PbtO2 with tissue saturation, multiple types of NIRS should be included via multimodal systemic- and neuro-monitoring, whose values then are incorporated into biosignatures linked to patient status and prognosis. Discussion on the abnormalities in tissue respiration due to brain trauma and how they affect the PbtO2 and NIRS neuromonitoring is given.

Taking Advantage of the Morpheein Behavior of Peroxiredoxin in Bionanotechnology.

Morpheeins are proteins that reversibly assemble into different oligomers, whose architectures are governed by conformational changes of the subunits. This property could be utilized in bionanotechnology where the building of nanometric and new high-ordered structures is required. By capitalizing on the adaptability of morpheeins to create patterned structures and exploiting their inborn affinity toward inorganic and living matter, "bottom-up" creation of nanostructures could be achieved using a single protein building block, which may be useful as such or as scaffolds for more complex materials. Peroxiredoxins represent the paradigm of a morpheein that can be applied to bionanotechnology. This review describes the structural and functional transitions that peroxiredoxins undergo to form high-order oligomers, e.g., rings, tubes, particles, and catenanes, and reports on the chemical and genetic engineering approaches to employ them in the generation of responsive nanostructures and nanodevices. The usefulness of the morpheeins' behavior is emphasized, supporting their use in future applications.

Ligand-Linked Association-Dissociation in Transport Proteins and Hormone Receptors.

Ligand-linked changes in the aggregation state of biological macromolecules occur and have importance in several physiological processes, e.g., the response of hormone receptors, cooperative ligand binding, and others. The mathematical formalisms that express the thermodynamics governing these processes are complex, as they are required to describe observations made under experimental conditions in which many parameters may be simultaneously varied. The description of the functional behaviour of proteins that present ligand-linked association-dissociation events must accommodate cases where both the binding stoichiometries and reaction mechanisms are variable. In this paper, we review some paradigmatic cases that cover different structural arrangements and binding modes, with special attention to the case of dissociating homodimeric transport proteins and receptors. Even though we cannot pretend to be comprehensive on the proteins presenting this behaviour, we believe that we can attempt to be comprehensive on the structural arrangements and thermodynamic properties of these systems, which fall into a limited set of possible types.

Control of Oxygen Affinity in Mammalian Hemoglobins: Implications for a System Biology Description of the Respiratory Properties of the Red Blood Cell.

Hemoglobin and myoglobin have been considered for a long time the paradigmatic model systems for protein function, to the point of being defined the "hydrogen atom[s] of biology". Given this privileged position and the huge amount of quantitative information available on these proteins, the red blood cell might appear as the model system and"hydrogen atom" of system biology. Indeed, since the red cell's main function is O2 transport by hemoglobin, the gap between the protein and the cell may appear quite small. Yet, a surprisingly large amount of detailed biochemical information is required for the modelization of the respiratory properties of the erythrocyte. This problem is compounded if modelization aims at uncovering or explaining evolutionarily selected functional properties of hemoglobin. The foremost difficulty lies in the fact that hemoglobins having different intrinsic properties and relatively ancient evolutionary divergence may behave similarly in the complex milieu of blood, whereas very similar hemoglobins sharing a substantial sequence similarity may present important functional differences because of the mutation of a few key residues. Thus, the functional properties of hemoglobin and blood may reflect more closely the recent environmental challenges than the remote evolutionary history of the animal. We summarize in this review the case of hemoglobins from mammals, in an attempt to provide a reasoned summary of their complexity that, we hope, may be of help to scientists interested in the quantitative exploration of the evolutionary physiology of respiration. Indeed the basis of a meaningful modelization of the red cell requires a large amount of information collected in painstaking and often forgotten studies of the biochemical properties of hemoglobin carried out over more than a century.

Ectopic suicide inhibition of thioredoxin glutathione reductase.

Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.

On the Measurement of Cooperativity and the Physico-Chemical Meaning of the Hill Coefficient.

Cooperative ligand binding is a fundamental property of many biological macromolecules, notably transport proteins, hormone receptors, and enzymes. Positive homotropic cooperativity, the form of cooperativity that has greatest physiological relevance, causes the ligand affinity to increase as ligation proceeds, thus increasing the steepness of the ligand-binding isotherm. The measurement of the extent of cooperativity has proven difficult, and the most commonly employed marker of cooperativity, the Hill coefficient, originates from a structural hypothesis that has long been disproved. However, a wealth of relevant biochemical data has been interpreted using the Hill coefficient and is being used in studies on evolution and comparative physiology. Even a cursory analysis of the pertinent literature shows that several authors tried to derive more sound biochemical information from the Hill coefficient, often unaware of each other. As a result, a perplexing array of equations interpreting the Hill coefficient is available in the literature, each responding to specific simplifications or assumptions. In this work, we summarize and try to order these attempts, and demonstrate that the Hill coefficient (i) provides a minimum estimate of the free energy of interaction, the other parameter used to measure cooperativity, and (ii) bears a robust statistical correlation to the population of incompletely saturated ligation intermediates. Our aim is to critically evaluate the different analyses that have been advanced to provide a physical meaning to the Hill coefficient, and possibly to select the most reliable ones to be used in comparative studies that may make use of the extensive but elusive information available in the literature.

Apixaban Interacts with Haemoglobin: Effects on Its Plasma Levels.

The direct oral anticoagulant apixaban (APX), a strong factor Xa inhibitor, binds also to plasma proteins, especially albumin, and minimally to α1-acid glycoprotein. Although APX can cross the red cell membrane, due to its chemical structure, and could bind to haemoglobin (Hb), no investigation was performed on this possible phenomenon that could affect the APX plasma concentration and thus its pharmacokinetics and pharmacodynamics. We addressed this issue by (1) measuring the levels of APX and haematological/biochemical parameters in 90 patients on APX therapy; (2) assessing the effect of APX on oxygen saturation curves of Hb; (3) testing the direct APX binding to Hb by fluorescence spectroscopy and a zinc-induced precipitation of Hb coupled to a reversed-phase high-performance liquid chromatography (HPLC)-based method; and (4) simulating in silico by molecular docking the APX interaction with human Hb. In a multivariable analysis, Hb was the only independent variable significantly and inversely associated in 90 patients with APX peak plasma level, at variance with patients treated with rivaroxaban (n = 86) and dabigatran (n = 34) therapy. APX causes a progressive left-shift of the oxygen dissociation curve of purified Hb solution, with a Kd ≅300 µM. Fluorescence- and HPLC-based assays concordantly showed that APX binds to Hb with a Kd ≅350 µM. Finally, docking simulations showed that APX can fit into in the central cavity of Hb. These findings support the hypothesis that APX does bind to Hb, which, due to its millimolar concentration in blood, can act as 'buffer' for the drug and consequently affect its free plasma level.

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Non-Allosteric Cooperativity in Hemoglobin.

Hemoglobin (Hb) is the prototypical example of a cooperative protein. Cooperativity of Hb is largely accounted for by the oxygen-linked allosteric interconversion between the T and R states/structures. Allostery is such a powerful explanation of Hb cooperativity that the possibility of cooperative events occurring within each allosteric conformation, in the absence of any quaternary structural change has usually been overlooked, and actually experiments specifically aimed at detecting nonallosteric cooperativity have usually failed to do so. However there are strong, but often neglected, theoretical reasons pointing to the presence of nonallosteric cooperativity under common experimental conditions, that have recently raised new interest and have been thoroughly re-investigated. Non-allosteric cooperativity within T state Hb has often been invoked to describe puzzling experimental data, either as an intrinsic property of the macromolecule or as a consequence of the binding of non-heme ligands. Few convincing pieces of evidence exist for the former hypothesis, whereas very strong proofs are available for effector-induced non-allosteric cooperativity in hemoglobin. Moreover, non-allosteric cooperativity in THb may explain some hitherto puzzling findings, e.g. the bi-exponential O2 release from THb observed by Q.H. Gibson in oxygen pulse experiments, the invariance of L4 found by K. Imai, the cooperative ligand binding by crystals of T state Hb Rotschild, and, possibly, the cooperativity observed in at least some mixed metal hybrid Hbs.

Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NH2 as a reliable strategy for antipseudomonal drugs.

Naturally occurring antimicrobial peptides (AMPs) hold promise as future therapeutics against multidrug resistant microorganisms. Recently, we have discovered that a derivative of the frog skin AMP esculentin-1a, Esc(1-21), is highly potent against both free living and biofilm forms of the bacterial pathogen Pseudomonas aeruginosa. However, bringing AMPs into clinics requires to overcome their low stability, high toxicity and inefficient delivery to the target site at high concentrations. Importantly, peptide conjugation to gold nanoparticles (AuNPs), which are among the most applied inorganic nanocarriers in biomedical sciences, represents a valuable strategy to solve these problems. Here we report that covalent conjugation of Esc(1-21) to soluble AuNPs [AuNPs@Esc(1-21)] via a poly(ethylene glycol) linker increased by ∼15-fold the activity of the free peptide against the motile and sessile forms of P. aeruginosa without being toxic to human keratinocytes. Furthermore, AuNPs@Esc(1-21) resulted to be significantly more resistant to proteolytic digestion and to disintegrate the bacterial membrane at very low concentration (5nM). Finally, we demonstrated for the first time the capability of peptide-coated AuNPs to display a wound healing activity on a keratinocytes monolayer. Overall, these findings suggest that our engineered AuNPs can serve as attractive novel biological-derived material for topical treatment of epithelial infections and healing of the injured tissue. STATEMENT OF SIGNIFICANCE: Despite conjugation of AMPs to AuNPs represents a worthwhile solution to face some limitations for their development as new therapeutics, only a very limited number of studies is available on peptide-coated AuNPs. Importantly, this is the first report showing that a covalent binding of a linear AMP via a poly(ethylene glycol) linker to AuNPs highly enhances antipseudomonal activity, preserving the same mode of action of the free peptide, without being harmful. Furthermore, AuNPs@Esc(1-21) are expected to accelerate recovery of an injured skin layer. All together, these findings suggest our peptide-coated AuNPs as attractive novel nanoscale formulation to treat bacterial infections and to heal the injured tissue.

Typical 2-Cys peroxiredoxins in human parasites: Several physiological roles for a potential chemotherapy target.

Peroxiredoxins (Prxs) are ubiquitary proteins able to play multiple physiological roles, that include thiol-dependent peroxidase, chaperone holdase, sensor of H2O2, regulator of H2O2-dependent signal cascades, and modulator of the immune response. Prxs have been found in a great number of human pathogens, both eukaryotes and prokaryotes. Gene knock-out studies demonstrated that Prxs are essential for the survival and virulence of at least some of the pathogens tested, making these proteins potential drug targets. However, the multiplicity of roles played by Prxs constitutes an unexpected obstacle to drug development. Indeed, selective inhibitors of some of the functions of Prxs are known (namely of the peroxidase and holdase functions) and are here reported. However, it is often unclear which function is the most relevant in each pathogen, hence which one is most desirable to inhibit. Indeed there are evidences that the main physiological role of Prxs may not be the same in different parasites. We here review which functions of Prxs have been demonstrated to be relevant in different human parasites, finding that the peroxidase and chaperone activities figure prominently, whereas other known functions of Prxs have rarely, if ever, been observed in parasites, or have largely escaped detection thus far.

One ring (or two) to hold them all ­ on the structure and function of protein nanotubes.

Understanding the structural determinants relevant to the formation of supramolecular assemblies of homo-oligomeric proteins is a traditional and central scope of structural biology. The knowledge thus gained is crucial both to infer their physiological function and to exploit their architecture for bionanomaterials design. Protein nanotubes made by one-dimensional arrays of homo-oligomers can be generated by either a commutative mechanism, yielding an 'open' structure (e.g. actin), or a noncommutative mechanism, whereby the final structure is formed by hierarchical self-assembly of intermediate 'closed' structures. Examples of the latter process are poorly described and the rules by which they assemble have not been unequivocally defined. We have collected and investigated examples of homo-oligomeric circular arrangements that form one-dimensional filaments of stacked rings by the noncommutative mechanism in vivo and in vitro. Based on their quaternary structure, circular arrangements of protein subunits can be subdivided into two groups that we term Rings of Dimers (e.g. peroxiredoxin and stable protein 1) and Dimers of Rings (e.g. thermosome/rosettasome), depending on the sub-structures that can be identified within the assembly (and, in some cases, populated in solution under selected experimental conditions). Structural analysis allowed us to identify the determinants by which ring-like molecular chaperones form filamentous-like assemblies and to formulate a novel hypothesis by which nanotube assembly, molecular chaperone activity and macromolecular crowding may be interconnected.

Selenocysteine robustness versus cysteine versatility: a hypothesis on the evolution of the moonlighting behaviour of peroxiredoxins.

Peroxiredoxins (Prxs) and glutathione peroxidases (Gpxs) provide the majority of peroxides reducing activity in the cytoplasm. Both are peroxidases but differences in the chemical mechanism of reduction of oxidative agents, as well as in the reactivity of the catalytically active residues, confer peculiar features on them. Ultimately, Gpx should be regarded as an efficient peroxides scavenger having a high-reactive selenocysteine (Sec) residue. Prx, by having a low pKa cysteine, is less efficient than Gpx in reduction of peroxides under physiological conditions, but the chemistry of the sulfur together with the peculiar structural arrangement of the active site, in typical Prxs, make it suitable to sense a redox environment and to switch-in-function so as to exert holdase activity under redox-stress conditions. The complex macromolecular assembly would have evolved the chaperone holdase function and the moonlighting behaviour typical of many Prxs.

Thioredoxin reductase and its inhibitors.

Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis.

Hemoglobin allostery: new views on old players.

Proteins are dynamic molecular machines whose structure and function are modulated by environmental perturbations and natural selection. Allosteric regulation, discovered in 1963 as a novel molecular mechanism of enzymatic adaptation [Monod, Changeux & Jacob (1963). J. Mol. Biol.6, 306-329], seems to be the leit motiv of enzymes and metabolic pathways, enabling fine and quick responses toward external perturbations. Hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the paradigmatic system to test the validity of the conformational selection mechanism, the conceptual innovation introduced by Monod, Wyman and Changeux. We present hereby the results of a comparative analysis of structure, function and thermodynamics of two extensively investigated hemoglobins: human HbA and trout HbI. They represent a unique and challenging comparison to test the general validity of the stereochemical model proposed by Perutz. Indeed both proteins are ideal for the purpose being very similar yet very different. In fact, T-HbI is a low-ligand-affinity cooperative tetrameric Hb, insensitive to all allosteric effectors. This remarkable feature, besides being physiologically sound, supports the stereochemical model, given that the six residues identified in HbA as responsible for the Bohr and the 2,3-di-phosphoglycerate effects are all mutated. Comparison of the three-dimensional structures of HbA and T-HbI allows unveiling the molecular mechanism whereby the latter has a lower O2 affinity. Moreover, the energetic balance sheet shows that the salt bridges breaking upon allosteric quaternary transition are important yet insufficient to account for the free energy of heme-heme interactions in both hemoglobins.

Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target.

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.