Stereospecific Binding Research Articles

Tefluthrin induced toxicities in zebrafish: Focusing on enantioselectivity

Tefluthrin is one of widely used chiral pyrethroid pesticides. The potential enantioselective risk posed by tefluthrin to the aquatic ecosystem is still unclear. In this study, the toxicity differences and corresponding mechanism of tefluthrin on zebrafish were investigated at the enantiomeric level. The results indicated that two tefluthrin enantiomers showed different acute toxicity, developmental toxicity and oxidative stress to zebrafish. The acute toxicity of (1R,3R)-tefluthrin was 130–176 fold as that of (1S,3S)-tefluthrin on zebrafish embryos, larvae and adults. (1R,3R)-Tefluthrin presented approximately 10, 3 and 2 times inhibition effect on the deformity rate, hatching rate and spontaneous movements on embryos as that of (1S,3S)-tefluthrin. Meanwhile, (1R,3R)-tefluthrin caused stronger oxidative stress on zebrafish embryo than (1S,3S)-tefluthrin. The molecular docking results revealed that there were stereospecific binding affinities between tefluthrin enantimers and sodium channel protein (Nav1.6), which may lead to acute toxicity differences. Transcriptome analysis showed that the two tefluthrin enantiomers markedly disturbed differential embryonic genes expression, thereby potentially causing the chronic enantioselective toxicity. The findings of the study reveal the toxicity differences and potential mechanism of tefluthrin enantiomers on zebrafish. These results also provides a foundation for a systematic evaluation of tefluthrin at enantiomer level.

Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.

The stereospecific interaction sites and target specificity of cGMP analogs in mouse cortex.

cGMP interactors play a role in several pathologies and may be targets for cGMP analog-based drugs, but the success of targeting depends on the biochemical stereospecificity between the cGMP-analog and the interactor. The stereospecificity between general cGMP analogs-or such that are selectivity-modified to obtain, for example, inhibitory actions on a specific target, like the cGMP-dependent protein kinase-have previously been investigated. However, the importance of stereospecificity for cGMP-analog binding to interactors is not known. We, therefore, applied affinity chromatography on mouse cortex proteins utilizing analogs with cyclic phosphate (8-AET-cGMP, 2-AH-cGMP, 2'-AHC-cGMP) and selectivity-modified analogs with sulfur-containing cyclic phosphorothioates (Rp/Sp-8-AET-cGMPS, Rp/Sp-2'-AHC-cGMPS) immobilized to agaroses. The results illustrate the cGMP analogs' stereospecific binding for PKG, PKA regulatory subunits and PKA catalytic subunits, PDEs, and EPAC2 and the involvement of these in various KEGG pathways. For the seven agaroses, PKG, PKA regulatory subunits, and PKA catalytic subunits were more prone to be enriched by 2-AH-, 8-AET-, Rp-8-AET-, and Sp-8-AET-cGMP, whereas PDEs and EPAC2 were more likely to be enriched by 2-AH-, Rp-2'-AHC-, and Rp-8-AET-cGMP. Our findings help elucidate the stereospecific-binding sites essential for the interaction between individual cGMP analogs and cGMP-binding proteins, as well as the cGMP analogs' target specificity, which are two crucial parameters in drug design.

Intrinsic Clearance and Metabolism Pathway of Fosthiazate in Rat and Cock Liver Microsomes: From Chiral Assessment View.

Chiral fosthiazate enters the organisms via environmental exposure and food web enrichment. Liver subcellular fractions of rats (RLM) and cocks (CLM) were prepared to explore the stereoselective metabolism of fosthiazate in vitro. The results indicated that fosthiazate exhibited different stereoselective metabolism behaviors in RLM and CLM. The clearance rate order of RLM to four fosthiazate stereoisomers was (1R,3R)-fosthiazate > (1S,3R)-fosthiazate > (1R,3S)-fosthiazate > (1S,3S)-fosthiazate. However, CLM showed a faster clearance rate to (1S,3S)-fosthiazate and (1S,3R)-fosthiazate than the other two stereoisomers. The molecular docking results revealed that the stereoselectivity was partially due to the stereospecific binding between fosthiazate stereoisomers and cytochrome P450 proteins. The main metabolism pathways of fosthiazate in RLM and CLM were oxidation and hydrolysis with five common metabolites including M299, M243, M227, M103, and M197 being identified by LC-TOF-MS/MS. The present study provides the accurate data on risk assessment of chiral fosthiazate.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

3,4-Dihydropyrimidin-2(1H)-ones as Antagonists of the Human A2B Adenosine Receptor: Optimization, Structure-Activity Relationship Studies, and Enantiospecific Recognition.

We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2(1H)-ones as A2BAR antagonists, an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A2BAR, with a number of potent and selective antagonists further confirmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure-activity relationship of this chemotype. The stereospecific binding modeled for this scaffold was confirmed by resolving the two most potent ligands [(±)-47, and (±)-38 Ki = 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the affinity on the corresponding (S)-eutomers (Ki = 6.30 and 11.10 nM, respectively). The assessment of the effect in representative cytochromes (CYP3A4 and CYP2D6) demonstrated insignificant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that this pair of compounds exhibits a pronounced antimetastatic effect.

Millisecond Conformational Dynamics of Skeletal Myosin II Power Stroke Studied by High-Speed Atomic Force Microscopy.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of Pi after the power stroke.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site

AbstractElectrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.

Dimethylphenylphosphine oxide coordinated trivalent rhenium featuring pyridylbenzazole chelation: Oxygen atom transfer kinetics, isomer preference, metal oxidation and computational analysis

The present work embodies outward oxygen atom transfer (OAT) reaction from pyridylbenzoxazole (L1) chelated oxorhenium(V) complex of type [ReOCl3(L1)], 1 towards oxophilic PMe2Ph resulting in the formation of reduced Re(III) derivative [Re(OPMe2Ph)Cl3(L1)], 4 containing oxidised phosphine trans to azole nitrogen. Another possible isomer (4a) having oxidised phosphine trans to pyridyl nitrogen has not been isolated. Structural elucidation of 4 reveals meridional disposition of three Cl atoms around the metal featuring distorted octahedral geometry. DFT analysis of isomers of type [Re(OPMe2Ph)Cl3(L1)] (4 and 4a) confirms isoenergetic nature in gas phase, however, stereospecific binding of L1 to Re(III) centre affording 4 as exclusive product is attributed to crystal packing force. 1 → 4 redox transformation has also been attended with the change in singlet → triplet spin state of the metal. Triplet state of 4 is more stable by ca. 15 kcal mol−1 compared to the singlet state in gas phase. Two electron paramagnetic nature (2.13 μB) in solid state affirms triplet ground state characterised with strong orbital coupling. Kinetic data for 1 → 4 transformation suggests associative pathway (ΔS≠ = ∼−36 J/K/mol) with no major structural reorganisation in the primary coordination sphere of the substrate and the reagent (ΔH≠ = ∼+10 kcal mol−1) to reach the transition state. Pyridylbenzthiazole (L2) chelated oxorhenium(V) complex of type [ReOCl3(L2)], 1b also reacts with PMe2Ph in a same manner to from [Re(OPMe2Ph)Cl3(L2)], 4b where the pyridyl nitrogen lies trans to phosphineoxide moiety unlike 4. The second order rate constants of OAT to PMe2Ph for [ReOCl3(L1)] are nearly two times higher than that of [ReOCl3(L2)] at all recorded temperatures due to difference in azole heteroatom electronegativity, ReVO bond length and ReVI/ReV reduction potential.

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.

Stereospecific modulation of dimeric rhodopsin.

The classic concept that GPCRs function as monomers has been challenged by the emerging evidence of GPCR dimerization and oligomerization. Rhodopsin (Rh) is the only GPCR whose native oligomeric arrangement was revealed by atomic force microscopy demonstrating that Rh exists as a dimer. However, the role of Rh dimerization in retinal physiology is currently unknown. In this study, we identified econazole and sulconazole, two small molecules that disrupt Rh dimer contacts, by implementing a cell-based high-throughput screening assay. Racemic mixtures of identified lead compounds were separated and tested for their stereospecific binding to Rh using UV-visible spectroscopy and intrinsic fluorescence of tryptophan (Trp) 265 after illumination. By following the changes in UV-visible spectra and Trp265 fluorescence in vitro, we found that binding of R-econazole modulates the formation of Meta III and quenches the intrinsic fluorescence of Trp265. In addition, electrophysiological ex vivo recording revealed that R-econazole slows photoresponse kinetics, whereas S-econazole decreased the sensitivity of rods without effecting the kinetics. Thus, this study contributes new methodology to identify compounds that disrupt the dimerization of GPCRs in general and validates the first active compounds that disrupt the Rh dimer specifically.-Getter, T., Gulati, S., Zimmerman, R., Chen, Y., Vinberg, F., Palczewski, K. Stereospecific modulation of dimeric rhodopsin.

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTSsugar) and one for nitrogen regulation (PTSNtr), that utilize proteins enzyme Isugar (EIsugar) and HPr, and enzyme INtr (EINtr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EINtr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EINtr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EINtr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EINtr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.

Comparative docking analysis of cholesterol analogs to ion channels to discriminate between stereospecific binding vs. stereospecific response

ABSTRACTCholesterol is a major component of the membrane and a key regulator of many ion channels. Multiple studies showed that cholesterol regulates ion channels in a stereospecific manner, with cholesterol but not its chiral isomers having a functional effect. This stereospecificity has been universally attributed to the specificity of cholesterol binding, with the assumption that only native cholesterol binds to the channels whereas its isomers do not. In this study, we challenge this paradigm by docking analyses of cholesterol and its chiral isomers to five ion channels whose response to cholesterol was shown to be stereospecific, Kir2.2, KirBac1.1, TRPV1, GABAA and BK. The analysis is performed using AutoDock Vina to predict the binding poses and energies of the sterols to the channels and identify amino acids interacting with the sterol molecules. We found that for every ion channel tested herein all three sterols showed similar binding poses and significant overlap in the set of the amino acids that comprise the predicted binding sites, along with similar energetic favorability to these overlapping sites. We also found, however, that specific orientations of the three sterols within the binding sites of the channels are distinct, so that a subset of the interacting amino acids is unique to each sterol. We propose therefore, that contrary to previous thought, stereospecific effects of cholesterol should be attributed not to the lack of binding of the stereoisomers but to specific, unique interactions between the cholesterol molecule and the residues within the binding sites of the channels.

Molecular insights of inhibition in sickle hemoglobin polymerization upon glutathionylation: hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulation-based approach.

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of βVal6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of βCys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of βVal6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia.

PKU mutation p.G46S prevents the stereospecific binding of l-phenylalanine to the dimer of human phenylalanine hydroxylase regulatory domain.

Mammalian phenylalanine hydroxylase (PAH) has a potential allosteric regulatory binding site for l‐phenylalanine (l‐Phe), in addition to its catalytic site. This arrangement is supported by a crystal structure of a homodimeric truncated form of the regulatory domain of human PAH (hPAH‐RD 1–118/19–118) [Patel D et al. (2016) Sci Rep doi: 10.1038/srep23748]. In this study, a fusion protein of the domain (MBP‐(pepXa)‐hPAH‐RD 1–120) was overexpressed and recovered in a metastable and soluble state, which allowed the isolation of a dimeric and a monomeric fusion protein. When cleaved from MBP, hPAH‐RD forms aggregates which are stereospecifically inhibited by l‐Phe (> 95%) at low physiological concentrations. Aggregation of the cleaved dimer of the mutant form hPAH‐G46S‐RD was not inhibited by l‐Phe, which is compatible with structurally/conformationally changed βαββαβ ACT domain folds in the mutant.

Differential Effects of Sterols on Ion Channels: Stereospecific Binding vs Stereospecific Response.

Numerous ion channels have been shown to be regulated by the level of membrane cholesterol, but the mechanisms responsible for these effects are still not well understood. The key question in the field is how to discriminate between the contributions of the two central mechanisms that might be responsible for the sensitivity of ion channels to cholesterol: specific sterol-protein interactions or regulation of channels by the bilayer physical properties. Comparative analysis of cholesterol and its isomers on the function of an ion channel is a powerful tool to achieve this goal. An increasing number of studies show that cholesterol regulates several types of ion channels in a stereospecific manner, suggesting an involvement of specific sterol-protein interactions. However in this chapter, we present evidence that the stereospecificity of cholesterol-ion channel interactions may be mediated, not by a lack of binding, as has been generally assumed, but by the specificity of the interaction, which results in a functional effect, in the case of native cholesterol, and a lack of functional effect, in the case of a cholesterol isomer. In other words, accumulating evidence suggests that the structural requirements of ion channel cholesterol-binding sites are lax, allowing chiral isomers of cholesterol to bind to the same site in a nonstereospecific way, but the ability of a sterol to confer a functional effect on the channel activity can still be stereospecific. This is an important distinction both conceptually and methodologically. Indeed, our analysis shows that the orientations of cholesterol and its chiral isomer ent-cholesterol within a hydrophobic binding pocket of Kir2.2 are significantly different, and we propose that this difference may underlie distinct functional outcomes.

Stereoselective binding of agonists to the β2-adrenergic receptor: insights into molecular details and thermodynamics from molecular dynamics simulations.

The β2-adrenergic receptor (β2-AR) is one of the most studied G-protein-coupled receptors. When interacting with ligand molecules, it exhibits a binding characteristic that is strongly dependent on ligand stereoconfiguration. In particular, many experimental and theoretical studies confirmed that stereoisomers of an important β2-AR agonist, fenoterol, are associated with diverse mechanisms of binding and activation of β2-AR. The objective of the present study was to explore the stereoselective binding of fenoterol to β2-AR through the application of an advanced computational methodology based on enhanced-sampling molecular dynamics simulations and potentials of interactions tailored to investigate the stereorecognition effects. The results remain in very good, quantitative agreement with the experimental data (measured in the context of ligand-receptor affinities and their dependence on the temperature), which provides an additional validation for the applied computational protocols. Additionally, our results contribute to the understanding of stereoselective agonist binding by β2-AR. Although the significant role of the N2936.55 residue is confirmed, we additionally show that stereorecognition does not depend solely on the N293-ligand interactions; the stereoselective effects rely on the co-operation of several residues located on both the 6th and 7th transmembrane domains and on extracellular loops. The magnitude and character of the contributions of these residues may be very diverse and result in either enhancing or reducing the stereoselective effects. The same is true when considering the enthalpic and entropic contributions to the binding free energies, which also are dependent on the ligand stereoconfiguration.

Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR.

There is growing experimental evidence that many proteins exhibit a tendency for (ultra)weak homo- or hetero- oligomerization interactions. With the development of paramagnetic relaxation enhancement NMR spectroscopy it has become possible to characterize weak complexes experimentally and even detect complexes with affinities in the 1–25 mM range. We present evidence for a weak complex between cytochrome c peroxidase (CcP) molecules. In a previous study, we attached nitroxide based spin labels at three positions on CcP with the intent of observing intramolecular PRE effects. However, several intermolecular PRE effects were also observed suggesting a weak self-association between CcP molecules. The CcP–CcP complex was characterized using paramagnetic NMR and protein docking. The interaction occurs between the surface that is also part of the stereo-specific binding site for its physiological partner, cytochrome c (Cc), and several small, positively charged patches on the “back” of CcP. The CcP–CcP complex is not a stereo-specific complex. It is a dynamic ensemble of orientations, characteristic of an encounter state. The contact areas resemble those observed for CcP molecules in crystals. The CcP–CcP complex formation competes with that of the CcP-Cc complex. However, the affinity for Cc is much larger and thus it is expected that, under physiological conditions, auto-inhibition will be limited.Graphical A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0035-z) contains supplementary material, which is available to authorized users.