Ligand Binding Properties Research Articles

Substitution of Arginine 120 in Human D-Amino Acid Oxidase Favors FAD-Binding and Nuclear Mistargeting.

The peroxisomal enzyme human D-amino acid oxidase (hDAAO) is attracting attention owing to its role in degrading D-serine, the main co-agonist of N-methyl D-aspartate receptors in brain, and its involvement in brain functions and diseases. Here, we focused on arginine 120, a residue located at the protein interface, 20 Å from the assumed second ligand-binding site, showing a different orientation of the side chain in the hDAAO-benzoate complex, and corresponding to Ser119 in rat DAAO, which is part of a putative nuclear translocation signal (NTS). By substituting Arg120 in hDAAO with a glutamate (to mimic the active NTS) or a leucine (to eliminate the positive charge) the protein conformation, thermal stability, and kinetic properties are slightly altered, while the dimeric structure and the ligand-binding properties are unchanged. The most relevant alteration in Arg120 variants is the strongest interaction with FAD. Nevertheless, the activity assayed at low D-serine and FAD concentrations (resembling physiological conditions) was quite similar for wild-type and Arg120 hDAAO variants. These results resemble the ones obtained substituting another residue located at the interface region (i.e., the W209R variant), indicating that substitutions at the monomer-monomer interface mainly affects the FAD binding in hDAAO. Indeed, U87 glioblastoma cells transiently transfected for hDAAO variants show that substitution of Arg120 favors mistargeting: the increase in cytosolic localization observed for the variants promotes nuclear targeting, especially for the R120E hDAAO, without affecting cell viability. Notably, mistargeting to the nucleus is an innate process as it is apparent for the wild-type hDAAO, too: whether such a process is related to specific pathologic processes is still unknown.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Recognizing five molecular ligand-binding sites with similar chemical structure.

Accurate identification of ligand-binding sites and discovering the protein-ligand interaction mechanism are important for understanding proteins' functions and designing new drugs. Meanwhile, accurate computational prediction and mechanism research are two grand challenges in proteomics. In this article, ligand-binding residues of five ligands (ATP, ADP, GTP, GDP, and NAD) are predicted as a group, due to their similar chemical structures and close biological function relations. The data set of binding sites by five ligands (ATP, ADP, GTP, GDP, and NAD) are collated from Biolip database. Then, five features, containing increment of diversity value, matrix scoring value, auto-covariance, secondary structure information, and surface accessibility information are used in binding site predictions. The support vector machine (SVM) model is used with the five features to predict ligand-binding sites. Finally, prediction results are tested by fivefold cross validation. Accuracy (Acc) of five ligands (ATP, ADP, GTP, GDP, and NAD) achieves 77.4%, 71.2%, 82.1%, 82.9%, and 85.3%, respectively; and Matthew correlation coefficient (MCC) of the above five ligands achieves 0.549, 0.424, 0.643, 0.659, and 0.702, respectively. The research result shows that for ligands with similar chemical structures, microenvironment of their binding sites and their sensitivities to features are similar, while, differences of their ligand-binding properties exist at the same time. © 2019 Wiley Periodicals, Inc.

A novel differential scanning fluorimetry analysis of a humanized anti-cocaine mAb and its ligand binding characteristics

An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinities. Here, we used differential scanning fluorimetry (DSF) to characterize the stability and ligand binding properties of this antibody and its cocaine-binding Fab fragment. The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic. However, by using the “rotor” dye, DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide), which measures the rotational restriction of the fluorescent dye (as opposed to the Sypro orange dye which measures the hydrophobicity of the dye microenvironment), a simple two state thermal denaturation transition that is stabilized by ligand binding was observed for the h2E2 mAb, enabling Boltzmann fitting and quantitative thermodynamic analysis of the DASPMI DSF mAb cocaine and metabolite binding data. The computed affinities were consistent with ligand binding affinities determined using other techniques. Thus, this novel DASPMI DSF method can simply, inexpensively, and very rapidly generate ligand binding constants for the h2E2 mAb, despite the presence of multiple, overlapping, thermally unfolding protein domains characteristic of all mAbs. This approach is likely applicable to other mAbs currently in use for many research and therapeutic applications.

Influence of pH and ionic strengths on bilirubin binding to sheep serum albumin

Despite great degree of similarity in the structure and conformation of different serum albumins, they often show marked difference in the ligand binding properties. These differences and similarities would be useful in understanding the molecular basis of ligand binding to serum albumins. In the present communication binding of bilirubin with sheep and bovine serum albumin have been studied by visible difference spectroscopy as a function of pH and ionic strength. The results suggest that the structure of bilirubin binding site in sheep serum albumin is similar to that in bovine serum albumin and that complex between sheep serum albumin and bilirubin is stabilized by electrostatic interactions. Keywords: Sheep serum albumin, Bovine serum albumin, Bilirubin binding, ionic strength, pH.

Neonicotinoid trapping by the FA1 site of human serum albumin.

Neonicotinoids are a widely used class of insecticides that target the acetylcholine recognition site of the nicotinic acetylcholine receptors in the central nervous system of insects. Although neonicotinoids display a high specificity for insects, their use has been recently debated since several studies led to the hypothesis that they may have adverse ecological effects and potential risks to mammals and even humans. Due to their hydrophobic nature, neonicotinoids need specific carriers to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key carrier of endogenous and exogenous compounds. The in silico docking and ligand binding properties of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam to HSA are here reported. Neonicotinoids bind to multiple fatty acid (FA) binding sites, preferentially to the FA1 pocket, with high affinity. Values of the dissociation equilibrium constant for neonicotinoid binding FA1 of HSA (i.e., calc Kn ) derived from in silico docking simulations (ranging between 3.9 × 10-5 and 6.3 × 10-4 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (i.e., exp Kn ; ranging between 2.1 × 10-5 and 6.9 × 10-5 M). Accounting for the HSA concentration in vivo (~7.5 10-4 M), values of Kn here determined suggest that the formation of the HSA:neonicotinoid complexes may occur in vivo. Therefore, HSA appears to be an important determinant for neonicotinoid transport and distribution to tissues and organs, particularly to the liver where they are metabolized.

Programmed Cell-Death by Ferroptosis: Antioxidants as Mitigators

Iron, the fourth most abundant element in the Earth’s crust, is vital in living organisms because of its diverse ligand-binding and electron-transfer properties. This ability of iron in the redox cycle as a ferrous ion enables it to react with H2O2, in the Fenton reaction, to produce a hydroxyl radical (•OH)—one of the reactive oxygen species (ROS) that cause deleterious oxidative damage to DNA, proteins, and membrane lipids. Ferroptosis is a non-apoptotic regulated cell death that is dependent on iron and reactive oxygen species (ROS) and is characterized by lipid peroxidation. It is triggered when the endogenous antioxidant status of the cell is compromised, leading to lipid ROS accumulation that is toxic and damaging to the membrane structure. Consequently, oxidative stress and the antioxidant levels of the cells are important modulators of lipid peroxidation that induce this novel form of cell death. Remedies capable of averting iron-dependent lipid peroxidation, therefore, are lipophilic antioxidants, including vitamin E, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1) and possibly potent bioactive polyphenols. Moreover, most of the enzymes and proteins that cascade or interact in the pathway of ferroptosis such as a subunit of the cystine/glutamate transporter xc− (SLC7A11), glutathione peroxidase 4 (GPX4), and the glutamate–cysteine ligase (GCLC) iron metabolism genes transferrin receptor 1 (TfR1) ferroportin, (Fpn) heme oxygenase 1 (HO-1) and ferritin are regulated by the antioxidant response element of the transcription factor, Nrf2. These, as well as other radical trapping antioxidants (RTAs), are discussed in the current review.

Expression profiles and ligand-binding properties of two odorant-binding proteins from Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

Abstract The ligand-binding function of odorant binding proteins (OBPs) that are expressed exclusively or enriched in antennae are well-studied, whilst the ligand binding properties of relatively low expressed OBPs in insect antennae are still unclear. Here, two low expressed GmolOBPs (namely GmolOBP12 and GmolOBP16) were cloned based on the antennal transcriptome of Grapholita molesta, and then their expression profiles and binding properties were investigated via real-time quantitative PCR (qRT-PCR) and fluorescence binding assays, respectively. Multiple sequence alignment and phylogenetic tree analyses displayed that both of GmolOBP12 and GmolOBP16 possessed the typical six-cysteine motifs unique to the classic OBPs subfamily and were classified as such. Although the abundance of transcripts of GmolOBP12 and GmolOBP16 were relatively low compared to that of many other OBPs in the antennal transcriptome of G. molesta, qRT-PCR results indicated that the transcript levels of GmolOBP12 and GmolOBP16 in antennae were significantly higher than those in other tissues. GmolOBP12 displayed higher expression level in male antennae than in female antennae, while the transcript level of GmolOBP16 in antennae was similar for both sexes. Both recombinant GmolOBP12 and GmolOBP16 exhibited strong binding affinities to the sex pheromone (Z)-8-dodecenyl alcohol and host-plant volatile pear ester. Besides, rGmolOBP12 showed outstanding binding affinities to (E)-2-hexenal, benzaldehyde, 1-hexanol, and (Z)-3-hexenyl acetate with Ki values of 4.21, 6.81, 4.68 and 4.68 μM, respectively. rGmolOBP16 had moderate binding abilities with hexanal, decanal, 1-hexanol, methyl myristate and benzonitrile. We speculated that GmolOBP12 may have dual functions in recognition of green leaf volatiles and sex pheromone components and GmolOBP16 may participate in the detection of host-plant volatiles in chemoreception.

Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands.

The heme-based and CO-responsive RcoM transcriptional regulators from Burkholderia xenovorans are known to display an extremely high affinity for CO while being insensitive to O2. We have quantitatively characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2 to establish the origin of these characteristics. Whereas the CO binding rates are similar to those of other heme-based sensor proteins, the dissociation rates are two to three orders of magnitude lower. The latter property is tuned by the yield of CO escape from the heme pocket after disruption of the heme-CO bond, as determined by ultrafast spectroscopy. For the full-length protein this yield is ∼0.5%, and for the isolated heme domain it is even lower, associated with correspondingly faster CO rebinding kinetics, leading to Kd values of 4 and 0.25 nM, respectively. These differences imply that the presence of the DNA-binding domain influences the ligand-binding properties of the heme domain, thus abolishing the observed quasi-irreversibility of CO binding to the isolated heme domain. RcoM-2 binds target DNA with high affinity (Kd < 2 nM) when CO is bound to the heme, and the presence of DNA also influences the heme-CO rebinding kinetics. The functional implications of our findings are discussed.

Characterization of DNA binding and ligand binding properties of the TetR family protein involved in regulation of dsz operon in Gordonia sp. IITR100

Gordonia sp. IITR100 is a biodesulfurizing bacterium which can metabolize dibenzothiophene (DBT) to 2 hydroxybiphenyl in four steps via the 4S pathway. The genes involved in the metabolism are present in the form of an operon, dszABC, which gets activated by a TetR family protein. Here, we report the detailed characterization of the DNA binding and ligand binding property of the TetR family protein. The protein was found to be conserved across other desulfurizing organisms. The protein was purified and was found to exist as dimer. The presence of ligand binding site was identified by docking studies and the structural changes in the protein upon ligand binding were determined by CD spectroscopy and tryptophan fluorescence. Further, it was determined that this protein binds to an imperfect palindromic DNA sequence present in the dsz promoter DNA. Binding to the DNA also changes conformation of the protein.

Ligand-dependent inequivalence of the α and β subunits of ferric human hemoglobin bound to haptoglobin

Haptoglobin (Hp) prevents extra-erythrocytic hemoglobin- (Hb-)mediated damage. Hp binds αβ dimers of Hb, displaying heme-based reactivity. Here, kinetics and thermodynamics of cyanide, thiocyanate and imidazole binding to ferric human Hb (Hb(III)), Hb(III) dimers complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively), and α(III) and β(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 °C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) with a simple behavior. In contrast, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) follows a two-step process, whereas ligand binding to α(III) and β(III) chains follows a simple behavior. However, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) mirror the ligand binding properties of the β(III) and α(III) chains, respectively. Values of kinetic and thermodynamic parameters for binding of ferric ligands to Hp1-1:Hb(III) and Hp2-2:Hb(III) match very well with those obtained for ligation of Hb(III) and α(III) and β(III) chains, confirming the ligand-dependent kinetic inequivalence of α(III) and β(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) on the other side for the rate-limiting step (likely referable to the dissociation of heme-coordinated H2O from the heme-Fe(III) atom) suggests a structural change(s) upon dimers to tetramer assembly in Hb(III).

Resonance Raman optical activity of the imidazole–Myoglobin complex: Titrating enhancement

AbstractResonance Raman optical activity (rROA) is a very powerful chiroptical technique for the investigation of bioactive protein cofactors in native conformation, due to its sensitivity towards any tiny conformational changes occurring at the chromophore. Twelve years ago, the rROA spectrum of myoglobin was published for the first time, revealing the strong potential of the technique for the selective study of the haem moiety. In this contribution, the use of rROA has been extended to the ligand binding properties of myoglobin via a combined approach of resonance Raman/ROA and ultraviolet‐visible absorption/electronic circular dichroism. The treatment of myoglobin with molar excess of imidazole in aqueous solution has led to the formation of a brilliant red pigment, known as imidazolylmet‐myoglobin, and revealed an interesting evolution of the visible Q bands, with the appearance of Qα at 534 nm. The use of the laser excitation of 532 nm, in resonance with the S0–S1 electronic transition (Q bands) of the sample, induced the selective Raman and ROA enhancement of certain haem vibrational modes, in a concentration‐dependent manner. Here, we highlight the sensitivity of rROA spectroscopy towards conformational changes of the haem chromophore upon interaction with the exogenous molecule, allowing us to distinguish between the bound and unligated form of the globin, a fundamental step towards the full understanding of the haem‐ligand binding process.

Ligand-binding properties and catalytic activity of the purified human 24-hydroxycholesterol 7α-hydroxylase, CYP39A1

Oxysterols are derivatives of cholesterol and biologically active molecules that are involved in a number of functions, including cholesterol homeostasis, immune response, embryogenic development and pathophysiology of neurodegenerative diseases. Enzymes catalyzing their synthesis and metabolism are of particular interest as potential or evaluated drug targets. Here we report for the first time biochemical analysis of purified human oxysterol 7α-hydroxylase selective for 24-hydroxycholesterol. Binding analyses indicated a tight binding of the oxysterols and estrone. Ligand screening revealed that CYP39A1 binds with high affinity antifungal drugs and prostate cancer drug galeterone (TOK-001). Site-directed mutagenesis of conserved Asn residue in the active site revealed its crucial role for protein folding and heme incorporation. Developed protocol for expression and purification enables further investigation of this hepatic enzyme as off-target in development of specific drugs targeting cytochrome P450 enzymes.

Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments.

Cytochrome c oxidase (C cO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven C cO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other C cOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical “macro” (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD–ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP‐r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure–function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.

Active-Site Druggability of Carbapenemases and Broad-Spectrum Inhibitor Discovery.

Serine and metallo-carbapenemases are a serious health concern due to their capability to hydrolyze nearly all β-lactam antibiotics. However, the molecular basis for their unique broad-spectrum substrate profile is poorly understood, particularly for serine carbapenemases, such as KPC-2. Using substrates and newly identified small molecules, we compared the ligand binding properties of KPC-2 with the noncarbapenemase CTX-M-14, both of which are Class A β-lactamases with highly similar active sites. Notably, compared to CTX-M-14, KPC-2 was more potently inhibited by hydrolyzed β-lactam products (product inhibition), as well as by a series of novel tetrazole-based inhibitors selected from molecular docking against CTX-M-14. Together with complex crystal structures, these data suggest that the KPC-2 active site has an enhanced ability to form favorable interactions with substrates and small molecule ligands due to its increased hydrophobicity and flexibility. Such properties are even more pronounced in metallo-carbapenemases, such as NDM-1, which was also inhibited by some of the novel tetrazole compounds, including one displaying comparable low μM affinities against both KPC-2 and NDM-1. Our results suggest that carbapenemase activity confers an evolutionary advantage on producers via a broad β-lactam substrate scope but also a mechanistic Achilles' heel that can be exploited for new inhibitor discovery. The complex structures demonstrate, for the first time, how noncovalent inhibitors can be engineered to simultaneously target both serine and metallo-carbapenemases. Despite the relatively modest activity of the current compounds, these studies also demonstrate that hydrolyzed products and tetrazole-based chemotypes can provide valuable starting points for broad-spectrum inhibitor discovery against carbapenemases.

The Dual Specific Hemolysin‐X is a Bioactive Natural Product that Forms Pores on the Surface of Erythrocytes

Hemolysins are natural cytotoxic agents that are capable of destabilizing plasma membrane structures of erythrocytes. We recently identified a plant hemolysin named Hemolysin‐X (HelyX) that showed dual specificity for cholesterol and certain serum glycoproteins, such as asialofetuin and fibrinogen. HelyX was isolated through two different approaches: protein purification protocols as well as organic solvent extraction. Ligand binding properties of HelyX were elucidated by inhibition assays using microtiter plates as well as spectrophotometer. HelyX demonstrated significantly fast kinetics (30–180 seconds) of lysis and ligand binding. Scanning electron microscopic imaging captured distinct pores on the surface of HelyX‐treated erythrocytes. Our results indicate that HelyX disintegrates mammalian red blood cells by forming pores on their surfaces.Support or Funding InformationMichigan Technological UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane β-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the α-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind α-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for α-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic α-DG non-binders such as laminin-α3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of α-DG, although it appears that this alone is insufficient to mediate significant α-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with α-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 μs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT1B and 5-HT2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT2B; in the absence of such interactions, the extracellular surface of the 5-HT1B is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT1 receptor family and manipulate the architecture of the extracellular ligand-binding pocket.

Construction of Fucoxanthin Vector Based on Binding of Whey Protein Isolate and Its Subsequent Complex Coacervation with Lysozyme.

In this study, a novel vector for fucoxanthin (FX) was constructed using the ligand-binding property of whey protein isolate and its subsequent heteroprotein complex coacervation with lysozyme. The results showed that FX could quench the intrinsic fluorescence of the whey protein isolate by a static mechanism, indicating that they could spontaneously form a nanocomplex through non-covalent interactions. Moreover, the structural and electrostatic properties of the resulting whey protein were different from those before the binding of FX, and this could be well explained by molecular dynamics simulation. The size and ζ-potential tests showed that when the whey protein isolate was combined with FX and then coacervated with lysozyme, the heteroprotein ratio and pH, which affect the coacervation process, also changed compared to those of the free whey protein isolate. FT-IR spectroscopy results showed that FX was successfully encapsulated into complex coacervates. In addition, the heteroprotein system exhibited a higher loading efficiency and also provided a better protection for FX in heating, storage, and simulated gastrointestinal environments.