Allosteric Proteins

The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRα)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRα ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptorinteracting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K d derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the 15 N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.

The allosteric effect is one of the most important processes in regulating the function of proteins, and the elucidation of this phenomenon plays a significant role in understanding emergent behaviors in biological regulation. In this process, a perturbation, generated by a ligand in a part of the macromolecule (the allosteric site), moves along this system and reaches a specific (active) site, dozens of Ångströms away, with a great efficiency. The dynamics of this perturbation in the macromolecule can model precisely the allosteric process. In this article, we will be studying the general characteristics of allostery, using a time-dependent quantum approach to obtain rules that apply to this kind of process. Considering the perturbation as a wave that moves within the molecular system, we will characterize the allosteric process with three of the properties of this wave in the active site: (1) t a , the characteristic time for reaching that site, (2) A a , the amplitude of the wave in this site, and (3) B a , its corresponding spectral broadening. These three parameters, together with the process mechanism and the perturbation efficiency in the process, can describe the phenomenon. One of the main purposes of this paper is to link the parameters t a , A a , and B a and the perturbation efficiency to the characteristics of the system. There is another fundamental process for life that has some characteristics similar to allostery: the light-harvesting (LH) process in photosynthesis. Here, as in allostery, two distant macromolecular sites are involvedtwo sites dozens of Ångströms away. In both processes, it is particularly important that the perturbation is distributed efficiently without dissipating in the infinite degrees of freedom within the macromolecule. The importance of considering quantum effects in the LH process is well documented in literature, and the quantum coherences are experimentally proven by time-dependent spectroscopic techniques. Given the existing similarities between these two processes in macromolecules, in this work, we suggest using Quantum Mechanics (QM) to study allostery.

In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. DHDPS is part of the diaminopimelate pathway leading to lysine, coupling (S)-aspartate--semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from methicillin-resistant Staphylococcus aureus, an important bacterial pathogen, are reported. The enzyme was crystallized in a number of forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.45 A resolution. The space group was P1 and the unit-cell parameters were a = 65.4, b = 67.6, c = 78.0 A ˚, = 90.1, = 68.9, = 72.3 . The crystal volume per protein weight (V M ) was 2.34 A ˚3 Da À1 , with an estimated solvent content of 47% for four monomers per asymmetric unit. The structure of the enzyme will help to guide the design of novel therapeutics against the methicillin-resistant S. aureus pathogen.

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the "off" and "on" allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to wellordered locations that should be easier to target.

... 31 Maugard , T. , Remaud - Simeon , M. , Petre , D. and Monsan , P. ( 1997 ) Tetrahedron, 14, 5185 – 5194 . ... 40 Jacobsen , EE , Hoff , BH , Riise Moen , A. and Anthonsen , T. ( 2003 ) Journal of Molecular Catalysis. B: Enzymatic, 21, 55 – 58 . ...

Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)hydroxycholesterol and its synthetic analogue SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, noncompetitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogues. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other.

HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an in silico and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis.

Background: Cytochrome P450 3A4 (CYP3A4) can bind several substrate molecules simultaneously and exhibits cooperativity. Results: Ligand binding in the active site is preceded by functionally important interactions at a distinct peripheral site. Conclusion: The mechanism of cooperativity involves a ligand-induced allosteric transition. Significance: Allosteric mechanism suggested by our results transforms the view of the grounds and significance of CYP3A4 cooperativity. The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may there...

p38a Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38a kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-a). The central role of p38a activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38a activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38a inhibitors.

Cascade reactions of amidines with nitroallylic acetates and α-bromonitroalkenes provide potentially bioactive imidazoles in good to excellent yields in most cases. While 2,4-disubstituted imidazol-5-yl acetates are formed in the first case, 2,4-disubstituted imidazoles, bearing no substituent at position 5, are the products in the second case. These two series of imidazoles, viz. 2,4,5-trisubstituted and 2,4-disubstituted, were screened for their activity against the protozoan parasite Trypanosoma cruzi which is responsible for Chagas disease. As many as three compounds were as active as the standard benznidazole and two others were 2-3-fold more active highlighting the potential of substituted imidazoles, easily accessible from nitroalkenes, as possible anti-parasitic agents.

The dynamics of biological polymers, including proteins, RNA, and DNA, occur in very high-dimensional spaces. Many naturally occurring polymers can navigate a vast phase space and rapidly find their lowest free energy (folded) state. Thus, although the search process is stochastic, it is not completely random. Instead, it is best described in terms of diffusion along a downhill energy landscape. In this context, there have been many efforts to use simplified representations of the energetics, for which the potential energy is chosen to be a relatively smooth function with a global minimum that corresponds to the folded state. That is, instead of including every type of physical interaction, the broad characteristics of the landscape are encoded in approximate energy functions. We describe a particular class of models, called structure-based models, that can be used to explore the diffusive properties of biomolecular folding and conformational rearrangements. These energy functions m...

G protein-coupled receptors constitute a large and functionally diverse family of transmembrane proteins. They are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways and are among the most targeted proteins in drug discovery. Recent advances in crystallization methods have permitted to resolve the molecular structure of several members of the family. This chapter focuses on the impact of these structures in the use of homology modeling techniques for building three-dimensional models of homologous G protein-coupled receptors, higher order oligomers, and their complexes with ligands and signaling proteins.

Bovine S100 G (calbindin D 9k , small Ca 2+-binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca 2+ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo-and Ca 2+-loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca 2+-loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N-and C-terminal parts containing the EF-hands I and II, respectively.

Nuclear receptors (NRs) form homo-and/or heterodimers as central scaffolds of multiprotein complexes, which activate or repress gene transcription to regulate development, homeostasis, and metabolism. Recent studies on NR quaternary structure reveal novel mechanisms of receptor dimerization, the existence of tetrameric chromatin-bound NRs, and previously unanticipated protein-protein/protein-DNA interactions.

The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.

In structure-taste relationships of sweet substances, conformational analysis is important for the definition of the "active conformation" which actually interacts with the sweet taste receptor. This paper describes the synthesis and taste of some isovanillyl derivatives having a rigid structure, which can mimick only one possible conformation of their flexible analogues. We report also the solid state conformation of a sulfur-containing sweet isovanillyl derivative which was established by X-ray analysis. The relationships between conformation and taste were studied by comparison with the current models of the sweet taste receptor and by principal components analysis (PCA) using geometrical descriptors, followed by factor and cluster analysis. A strong correlation was proven between taste and geometry, which is able to explain the taste of rigid derivatives as well as the difference of taste between the two enantiomers of the same compound.

The m opioid receptor (mOR), the principal target to control pain, belongs to the G protein-coupled receptors (GPCRs) family, one of the most highlighted protein families due to their importance as therapeutic targets. The conformational flexibility of GPCRs is one of their essential characteristics as they take part in ligand recognition and subsequent activation or inactivation mechanisms. It is assessed that the intrinsic mechanical properties of the mOR, more specifically its particular flexibility behavior, would facilitate the accomplishment of specific biological functions, at least in their first steps, even in the absence of a ligand or any chemical species usually present in its biological environment. The study of the mechanical properties of the mOR would thus bring some indications regarding the highly efficient ability of the mOR to transduce cellular message. We therefore investigate the intrinsic flexibility of the mOR in its apo-form using all-atom Molecular Dynamics simulations at the sub-microsecond time scale. We particularly consider the mOR embedded in a simplified membrane model without specific ions, particular lipids, such as cholesterol moieties, or any other chemical species that could affect the flexibility of the mOR. Our analyses highlighted an important local effect due to the various bendability of the helices resulting in a diversity of shape and volume sizes adopted by the mOR binding site. Such property explains why the mOR can interact with ligands presenting highly diverse structural geometry. By investigating the topology of the mOR binding site, a conformational global effect is depicted: the correlation between the motional modes of the extra-and intracellular parts of mOR on one hand, along with a clear rigidity of the central mOR domain on the other

The µ opioid receptor (µOR), the principal target to control pain, belongs to the G protein-coupled receptors (GPCRs) family, one of the most highlighted protein families due to their importance as therapeutic targets. The conformational flexibility of GPCRs is one of their essential characteristics as they take part in ligand recognition and subsequent activation or inactivation mechanisms. It is assessed that the intrinsic mechanical properties of the µOR, more specifically its particular flexibility behavior, would facilitate the accomplishment of specific biological functions, at least in their first steps, even in the absence of a ligand or any chemical species usually present in its biological environment. The study of the mechanical properties of the µOR would thus bring some indications regarding the highly efficient ability of the µOR to transduce cellular message. We therefore investigate the intrinsic flexibility of the µOR in its apo-form using all-atom Molecular Dynamic...

Proteins have evolved to use water to help guide folding. A physically motivated, nonpairwise-additive model of water-mediated interactions added to a protein structure prediction Hamiltonian yields marked improvement in the quality of structure prediction for larger proteins. Free energy profile analysis suggests that long-range water-mediated potentials guide folding and smooth the underlying folding funnel. Analyzing simulation trajectories gives direct evidence that water-mediated interactions facilitate native-like packing of supersecondary structural elements. Long-range pairing of hydrophilic groups is an integral part of protein architecture. Specific water-mediated interactions are a universal feature of biomolecular recognition landscapes in both folding and binding.

Plant adaptation in variable soil nitrate concentrations involves sophisticated signaling and transport systems that modulate a variety of physiological and developmental responses. However, we know very little about their molecular mechanisms. It has recently been reported that many of these responses are regulated by a transceptor NRT1.1, a transporter cum receptor of nitrate signaling. NRT1.1 displays dual-affinity modes of nitrate binding and establishes phosphorylated/non-phosphorylated states at the amino acid residue threonine 101 in response to fluctuating nitrate concentrations. Here we report that intrinsic structural asymmetries between the protomers of the homodimer NRT1.1 provide a functional basis for having dual-affinity modes of nitrate binding and play a pivotal role for the phosphorylation switch. Nitrate-triggered local conformational changes facilitate allosteric communications between the nitrate binding and the phosphorylation site in one protomer, but such communications are impeded in the other. Structural analysis therefore suggests the functional relevance of NRT1.1 interprotomer asymmetries.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homologue, we have now solved the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer, and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37 amino acids extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [3H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding ...

Recently, we have reported the syntheses and antiproliferative activities of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives on melanoma cells. As a continuous work for antiproliferative agents in melanoma, here we report the synthesis of conformationally rigid analogs, phenyl-6,8-dihydropyrazolo[3,4-b][1,4]diazepin-7(1H)-one derivatives7a-g, 8a-o and their antiproliferative activities against A375P melanoma cell line and U937 hematopoietic cell line. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, N-(3-(1-benzyl-7-oxo-1,6, 7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-4-chloro-3-(trifluoro methyl)benzamide-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl) phenyl) ureido)-2-methylbenzamide (7b) exhibited potent activities (GI 50 = 0.43 lM and 0.06 lM) on both cell lines. It has been further confirmed to be a potent and selective Raf kinases inhibitor and also mild inhibitor of PI3Ka.

The hub protein LC8, interacts with over 100 binding partners in the cell and participates in a range of cell activities including mitosis and viral interactions. LC8 is a symmetric homodimer with two identical binding grooves that typically bind to intrinsically disordered (ID) regions of client proteins, linking them together. Recent experiments have revealed significant cooperativity in LC8: Isothermal titration calorimetry (ITC) experiments demonstrate a significant change in the affinity of the second binding event-as well as heterogeneous enthalpy/entropy signatures for different peptides. In order to investigate the atomic resolution mechanisms of cooperativity in these systems, we examine multiple LC8 bound to different client peptides in three different binding conditions-apo, singly bound, and doubly bound-using a series of μs-scale molecular dynamics (MD) simulations. We compare dynamics and fluctuations across the different conditions and for peptides with varying binding properties to elucidate the cooperativity signals in these systems and find that local fluctuations support a range of cooperative behaviors among the peptides.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the β1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor–Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the...

Allostery can be viewed as an effect of binding at one site of the protein to a second, often significantly distant functional site, enabling regulation of the protein function. In majority of cases, the molecular mechanisms and networks mediating distant communications that give rise to allostery are poorly understood. We have developed a rigidity-transmission allostery (RTA) algorithm, a computational method based on mathematical rigidity theory. Starting with an X-ray crystal structure or an ensemble of three dimensional snapshots, we model the protein as a constraint network (graph) consisting of vertices (atoms) and edges (i.e., constraints including covalent bonds, electrostatic bonds, hydrogen bonds, and hydrophobic contacts).

Discovery and development of the selective serotonin reuptake inhibitors mark a milestone in neuro-pharmacology. Drugs from this class alter the functioning of the serotonin system by the potentiation of serotonin through the negative allosteric modulation of its neuronal uptake by the human serotonin transporter. Selective serotonin reuptake inhibitors show few side effects compared to those caused by traditional antidepressants and they vary in the binding interactions formed during binding. Gener-ally, their binding involves three specific regions of the drug structures, each participating in vital inter-actions, such as salt bridge formation and additional hydrophobic interactions with conserved resi-dues in the central binding site of the target protein. Side effects, however, such as the initial lack of response to treatment, or drowsiness, nausea, and sexual dysfunction occasionally may arise. Addi-tional binding studies, furthermore, highlighted the importance of enantioselectivity in the binding of these compounds, raising concerns about the beneficial application of racemate mixtures of some of these compounds. Therefore, additional characterisation of binding and further structural improve-ment of this class of drugs is necessary. The recently synthesized sertraline salts, and functional deriv-atives of fluoxetine and citalopram show promising results in delivering antidepressant activity as well as in effectively overcoming anorexigenic side-effects in rodent models. Hence, despite certain non-desired effects associated with selective serotonin reuptake inhibitor applications, this class of drugs is considered as first-line medication in the management of major depression, and is carrying an excel-lent potential for the development and refinement of the currently available and novel antidepressant therapies.

The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRα)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRα ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptorinteracting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K d derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the 15 N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.

Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of L-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C 4 organic acids in Arabidopsis mitochondrial metabolism.

Octahydro-1H-indole-2-carboxylic acid is a key starting material for the synthesis of Perindopril and Trandolapril. A rapid, economical, simple, sensitive and reliable stability-indicating reverse phase HPLC method developed and validated for the quantitative determination of all isomers related to Octahydro-1H-indole-2-carboxylic acid and its related substances. The compound is non chromophoric, it has three chiral centers and there is a possibility of four pairs of enantiomers. Refractive index detector was used for the quantification of all its isomers. Optimized mobile phase is 10 mM potassium phosphate buffer with pH-3.0. The C18 column (Inertsil ODS-4, 250 mm×4.6 mm×5 µm) is used as the stationary phase with a mobile phase flow rate of 1.5 mLmin-1 and column temperature maintained at 35°C. The developed method was validated as per ICH guidelines for Accuracy, linearity, range, precision, ruggedness, robustness, solution stability, limit of quantification, and limit of detection. A linear range from LOQ to 150% performed for the analyte and its three isomers. The correlation coefficient obtained for all the isomers was more than 0.999. The recoveries obtained for all isomers were found in between 93.9% and 107.9%. The detection limit for all the isomers were about 0.006 mg/mL and the quantification limits were in between 0.022 mg/mL to 0.024 mg/mL respectively. The proposed method can be successfully applied for the quantification of the three isomers in Octahydro-1H-indole-2-carboxylic acid and provides a simple and cost effective quality control tool for routine analysis.

Erythrocyte pyruvate kinase (PK-R) deficiency is the most frequent cause of hereditary hemolytic anemia due to defective glycolysis. We report the molecular basis underlying PK-R deficiency in 28 patients from 26 unrelated families. Twenty-four different point mutations were detected in PKLR, 14 of which are reported here for the first time. Of these novel mutations, 12 encoded an amino acid substitution: Ile90Asn,

Cascade reactions of amidines with nitroallylic acetates and α-bromonitroalkenes provide potentially bioactive imidazoles in good to excellent yields in most cases. While 2,4-disubstituted imidazol-5-yl acetates are formed in the first case, 2,4-disubstituted imidazoles, bearing no substituent at position 5, are the products in the second case. These two series of imidazoles, viz. 2,4,5-trisubstituted and 2,4-disubstituted, were screened for their activity against the protozoan parasite Trypanosoma cruzi which is responsible for Chagas disease. As many as three compounds were as active as the standard benznidazole and two others were 2-3-fold more active highlighting the potential of substituted imidazoles, easily accessible from nitroalkenes, as possible anti-parasitic agents.

Cascade reactions of amidines with nitroallylic acetates and α-bromonitroalkenes provide potentially bioactive imidazoles in good to excellent yields in most cases. While 2,4-disubstituted imidazol-5-yl acetates are formed in the first case, 2,4-disubstituted imidazoles, bearing no substituent at position 5, are the products in the second case. These two series of imidazoles, viz. 2,4,5-trisubstituted and 2,4-disubstituted, were screened for their activity against the protozoan parasite Trypanosoma cruzi which is responsible for Chagas disease. As many as three compounds were as active as the standard benznidazole and two others were 2-3-fold more active highlighting the potential of substituted imidazoles, easily accessible from nitroalkenes, as possible anti-parasitic agents.

The long-held views on lock-and-key versus induced fit in binding arose from the notion that a protein exists in a single, most stable conformation, dictated by its sequence. However, in solution proteins exist in a range of conformations, which may be described by statistical mechanical laws and their populations follow statistical distributions. Upon binding, the equilibrium will shift in favor of the bound conformation from the ensemble of conformations around the bottom of the folding funnel. Hence here we extend the implications and the usefulness of the folding funnel concept to explain fundamental binding mechanisms.

Crystallographic and mutagenesis studies have unravelled the general features of the allosteric transition mechanism in pyruvate kinase. The enzyme displays a dramatic conformational change in going from the T-to the R-state. All three domains forming each subunit of the tetrameric enzyme undergo simultaneous and concerted rotations, in such a way that all subunit and domain interfaces are modified. This mechanism is unprecedented since in all tetrameric allosteric enzymes, characterised at atomic resolution, at least one of the domain or subunit interfaces remains unchanged on the T-to R-state transition. The molecular mechanism of allosteric regulation here proposed provides a rationale for the effect of single site mutations observed in the human erythrocyte pyruvate kinase associated with a congenital anaemia.

Some novel 3-phenyl-2-[(E)-2-phenylethenyl]-3,4-dihydroquinazolin-4-one derivatives possessing para-sulfonamides groups on the phenyl ring of the 2-phenylethenyl moiety have been synthesized and their COX-2 inhibitory activity evaluated. The stuctures of the synthesized compounds were confirmed on the basis of FT-IR, 1 H-NMR, 13 C-NMR and mass spectral data. The COX-2 inhibition screening assay revealed that 4-[(E)-2-{3-(4methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide had a maximum COX-2 inhibition (47.1%), at a concentration of 20 μM.

Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5′-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligan...

Contemporary protein structure is a result of the trade off between the laws of physics and the evolutionary selection. The polymer nature of proteins played a decisive role in establishing the basic structural and functional units of soluble proteins. We discuss how these elementary building blocks work in the hierarchy of protein domain structure, co-translational folding, as well as in enzymatic activity and molecular interactions. Next, we consider modulators of the protein function, such as intermolecular interactions, disorder-to-order transitions, and allosteric signaling, acting via interference with the protein's structural dynamics. We also discuss the post-translational modifications, which is a complementary intricate mechanism evolved for regulation of protein functions and interactions. In conclusion, we assess an anticipated contribution of discussed topics to the future advancements in the field.

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