PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems

Summary PEG has been activated using epoxy-oxirane, epichlorohydrin and periodate based reactions. The coupling to activated PEG of several protein ligands of different sizes was investigated. Glutathione, trypsin inhibitor, Protein A and anti-BSA have been bound to PEG and used to increase the selectivity of protein separation in aqueous two-phase systems.     

Light-induced activation of an inert surface for covalent immobilization of a protein ligand.

A simple and mild procedure is developed for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage. Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction. The photochemical step is carried out under dry conditions to exclude the possibility of undesirable reactions between the solvent and the highly reactive nitrene. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.     

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.     

Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.     

Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation.

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP.     

Structural basis for ligand recognition and activation of RAGE

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor involved in?inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling events upon binding of a variety of ligands, such as glycated proteins, amyloid-β, HMGB1, and S100 proteins. The X-ray crystal structure of the VC1 ligand-binding region of the human RAGE ectodomain was determined at 1.85?? resolution. The VC1 ligand-binding surface was mapped onto the structure from titrations with S100B monitored by heteronuclear NMR spectroscopy. These NMR chemical shift perturbations were used as input for restrained docking calculations to generate a model for the VC1-S100B complex. Together, the arrangement of VC1 molecules in the crystal and complementary biochemical studies suggest a role for self-association in RAGE function. Our results enhance understanding of the functional outcomes of S100 protein binding to RAGE and provide insight into mechanistic models for how the receptor is activated.     

G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.     

Molecular insights into ligand recognition and G protein coupling of the neuromodulatory orphan receptor GPR139

Dear Editor, The G protein-coupled receptor GPR139 is involved in neuro-modulation,and one of its agonists is in clinical trials for the treatment of cognitive ...     

New PEG2 type polyethylene glycol derivatives for protein modification

Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF. © Rapid Science Ltd. 1998     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.     

Molecular basis for ligand activation of the human KCNQ2 channel

The voltage-gated potassium channel KCNQ2 is responsible for M-current in neurons and is an important drug target to treat epilepsy, pain and several other diseases related to neuronal hyper-excitability. A list of synthetic compounds have been developed to directly activate KCNQ2, yet our knowledge of their activation mechanism is limited, due to lack of high-resolution structures. Here, we report cryo-electron microscopy (cryo-EM) structures of the human KCNQ2 determined in apo state and in complex with two activators, ztz240 or retigabine, which activate KCNQ2 through different mechanisms. The activator-bound structures, along with electrophysiology analysis, reveal that ztz240 binds at the voltage-sensing domain and directly stabilizes it at the activated state, whereas retigabine binds at the pore domain and activates the channel by an allosteric modulation. By accurately defining ligand-binding sites, these KCNQ2 structures not only reveal different ligand recognition and activation mechanisms, but also provide a structural basis for drug optimization and design.Subject terms: Cryoelectron microscopy, Mechanisms of disease     

Protein A--a new ligand for human C-reactive protein

Phosphorylcholine (PC) is a classical ligand of C-reactive protein (CRP), a clinically important acute phase protein. In search of new ligands, CRPs were affinity-purified from several pathological samples, which exhibited distinct molecular variants induced in different diseases. Both glycosylated and non-glycosylated CRPs showed calcium-independent differential-binding to Staphylococcus aureus cell-surface Protein A. CRP possesses separate binding sites for Protein A and PC with different binding constants. We have demonstrated that Protein A is another ligand in addition to PC establishing an extended definition of CRP. Protein A binding may impart immunomodulatory roles of CRP in combating microorganisms or other foreign materials.     

NMR dynamic studies suggest that allosteric activation regulates ligand binding in chicken liver bile acid-binding protein

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.     

Manipulation of ligand binding affinity by exploitation of conformational coupling

Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).