A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates. Copyright (C) 2002 John Wiley Sons, Ltd.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Methods for prediction of peptide binding to MHC molecules: A comparative study

Background: A variety of methods for prediction of peptide binding to major histocompatibility complex (MHC) have been proposed. These methods are based on binding motifs, binding matrices, hidden Markov models (HMM), or artificial neural networks (ANN). There has been little prior work on the comparative analysis of these methods. Materials and Methods: We performed a comparison of the performance of six methods applied to the prediction of two human MHC class I molecules, including binding matrices and motifs, ANNs, and HMMs. Results: The selection of the optimal prediction method depends on the amount of available data (the number of peptides of known binding affinity to the MHC molecule of interest), the biases in the data set and the intended purpose of the prediction (screening of a single protein versus mass screening). When little or no peptide data are available, binding motifs are the most useful alternative to random guessing or use of a complete overlapping set of peptides for selection of candidate binders. As the number of known peptide binders increases, binding matrices and HMM become more useful predictors. ANN and HMM are the predictive methods of choice for MHC alleles with more than 100 known binding peptides. Conclusion: The ability of bioinformatic methods to reliably predict MHC binding peptides, and thereby potential T-cell epitopes, has major implications for clinical immunology, particularly in the area of vaccine design.

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)(3)(OH) centres

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a (Fe2S2)-S-III cluster in place. In contrast, the other proteins are isolated in colourless forms, although a (Fe2S2)-S-III cluster may be assembled in the C42G protein via incubation with Fe-III and sulfide. The four mutant proteins were isolated as stable mononuclear Hg-II forms which were converted to unstable mononuclear Fe-III preparations that contain both holo and apo protein. The Fe-III systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe-III form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe-III form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-0020355-1.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6’) common to both the glycine receptor (GlyR) and GABAA receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter and the main pore blocker binding site. Recent studies have suggested that the GlyRs and GABAARs have divergent open state pore structures at the 6’ position. When both the human a1T6’C homomeric GlyR and the rat a1T6’Cb1T6’C heteromeric GABAAR were expressed in HEK293 cells, their 6’ residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidising agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABAAR pore structure at the 6’ level may vary between different expression systems.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Subtype-selective noncompetitive or competitive inhibition of human alpha(1)-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)- over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B-max) for human alpha(1B)-ARs without changing affinity (K-D), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [H-3] inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Man alpha 1-3[Man alpha 1-6]Man, which is a known ligand for jArtinM (Jeyaprakash et al, 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form. (C) 2010 Elsevier B V. All rights reserved

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Evaluation of the Binding Properties of Maghemite Nanoparticle Surface-Coated with Meso-2-3-Dimercaptosuccinic Acid to Serum Albumin

In this study the interaction between magnetic nanoparticles (MNPs) surface-coated with meso-2,3-dimercaptosuccinic acid (DMSA) with both bovine serum albumin (BSA) and human serum albumin (HSA) was investigated. The binding of the MNP-DMSA was probed by the fluorescence quenching of the BSA and HSA tryptophan residue. Magnetic resonance and light microscopy analyses were carried out in in vivo tests using female Swiss mice. The binding constants (K(b)) and the complex stoichiometries (n) indicate that MNP-DMSA/BSA and MNP-DMSA/HSA complexes have low association profiles. After five minutes following intravenous injection of MNP-DMSA into mice`s blood stream we found the lung firstly target by the MNP-DMSA, followed by the liver in a latter stage. This finding suggests that the nanoparticle`s DMSA-coating process probably hides the thiol group, through which albumin usually binds. This indicates that biocompatible MNP-DMSA is a very promising material system to be used as a drug delivery system (DDS), primarily for lung cancer treatment.