Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

Mass spectrometric investigation of the DNA-binding properties of an anthracycline with two trisaccharide chains

Cosmomycin D (CosD) is an anthracycline that has two trisaccharide chains linked to its ring system. Gel electrophoresis showed that CosD formed stable complexes with plasmid DNA under conditions where daunorubicin (Dn) and doxorubicin (Dx) dissociated to some extent during the experiments. The footprint and stability of CosD complexed with 10- and 16 trier DNA was investigated using several applications of electrospray ionisation mass spectrometry (ESI-MS). ESI-MS binding profiles showed that fewer CosD molecules bound to the sequences than Dn or Dx. In agreement with this, ESI-MS analysis of nuclease digestion products of the complexes showed that CosD protected the DNA to a greater extent than Dn or Dx. In tandem MS experiments, all CosD-DNA complexes were more stable than Dn- and Dx-DNA complexes. These results Support that CosD binds more tightly to DNA and exerts a larger footprint than ESI-MS investigations of the binding properties of CosD Could be carried out rapidly and using only small amounts of sample. (C) 2008 Elsevier Inc. All rights reserved.

Metal binding selectivity of oxa-aza macrocyclic ligand: a DFT study of first- and second-row transition metal for four coordination systems

A detailed theoretical study of the 1,7,1l,17-tetraoxa-2,6,12,16-tetraaza-cycloeicosane ligand ([20]AneN(4)O(4)) coordinated to Fe2+, Co2+, Ni2+, Ru2+, Rh2+, and Pd2+ transition metal ions was carried out with the B3LYP method. Two different cases were performed: when nitrogen is the donor atom (1a (q) ) and also with the oxygen as the donor atom (1b (q) ). For all the cases performed in this study 1a (q) structures were always more stable than the 1b (q) ones. Considering each row is possible to see that the energy increases with the increase of the atomic number. The M2+ cation binding energies for the 1a (q) complexes increase with the following order: Fe2+ < Ru2+ < Co2+ < Ni2+ < Rh2+ < Pd2+.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Falcarinol is a covalent cannabinoid CB1 receptor antagonist and induces pro-allergic effects in skin

The skin irritant polyyne falcarinol (panaxynol, carotatoxin) is found in carrots, parsley, celery, and in the medicinal plant Panax ginseng. In our ongoing search for new cannabinoid (CB) receptor ligands we have isolated falcarinol from the endemic Sardinian plant Seseli praecox. We show that falcarinol exhibits binding affinity to both human CB receptors but selectively alkylates the anandamide binding site in the CB(1) receptor (K(i)=594nM), acting as covalent inverse agonist in CB(1) receptor-transfected CHO cells. Given the inherent instability of purified falcarinol we repeatedly isolated this compound for biological characterization and one new polyyne was characterized. In human HaCaT keratinocytes falcarinol increased the expression of the pro-allergic chemokines IL-8 and CCL2/MCP-1 in a CB(1) receptor-dependent manner. Moreover, falcarinol inhibited the effects of anandamide on TNF-alpha stimulated keratinocytes. In vivo, falcarinol strongly aggravated histamine-induced oedema reactions in skin prick tests. Both effects were also obtained with the CB(1) receptor inverse agonist rimonabant, thus indicating the potential role of the CB(1) receptor in skin immunopharmacology. Our data suggest anti-allergic effects of anandamide and that falcarinol-associated dermatitis is due to antagonism of the CB(1) receptor in keratinocytes, leading to increased chemokine expression and aggravation of histamine action.

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.

Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABA receptors

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of alpha(1)S205C and alpha(1)T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (-NCS). We also provide new data that identify alpha(1)H101C and alpha(1)N102C as exclusive sites of the reaction of a diazepam derivative where the -Cl atom is replaced by a -NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of alpha(1)beta(2)gamma(2) receptors.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

A conserved motif at the 3$\sp\prime$ end of genomic RNA of mouse hepatitis virus required for host protein binding and viral RNA replication

The initial step in coronavirus-mouse hepatitis virus (MHV) replication is the synthesis of negative strand RNA from a positive strand genomic RNA template. Our approach to studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the protein(s) which recognizes these signals at the 3$\sp\prime$ end of genomic RNA of MHV. To determine whether host cellular and/or virus-specific proteins interact with the 3$\sp\prime$ end of the coronavirus genome, an RNase T$\sb1$ protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from either mock- or MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. A conserved 11 nucleotide sequence UGAAUGAAGUU at nucleotide positions 36 to 26 from the 3$\sp\prime$ end of genomic RNA was identified to be responsible for the specific binding of host proteins, by using a series of RNA probes with deletions and mutations in this region. The RNA probe containing the 11 nucleotide sequence bound approximately four host cellular proteins with a highly labeled 120 kDa and three minor species with sizes of 103, 81 and 55 kDa, assayed by UV-induced covalent cross-linking. Mutation of the 11 nucleotide motif strongly inhibited cellular protein binding, and decreased the amount of the 103 and 81 kDa proteins in the complex to undetectable levels and strongly reduced the binding of the 120 kDa protein. Less extensive mutations within this 11 nucleotide motif resulted in variable decreases in RNA-protein complex formation depending on each probe tested. The RNA-protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable to those observed with extracts from uninfected cells.^ To investigate the possible role of this 3$\sp\prime$ protein binding element in viral RNA replication in vivo, defective interfering RNA molecules with complete or partial mutations of the 11 nucleotide conserved sequence were transcribed in vitro, transfected to host 17Cl-1 cells in the presence of helper virus MHV-JHM and analyzed by agarose gel electrophoresis, competitive RT-PCR and direct sequencing of the RT-PCR products. Both negative strand synthesis and positive strand replication of DI RNA were affected by mutation that disrupts RNA-protein complex formation, even though the 11 mutated nucleotides were converted to wild type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred 5.5 to 7.5 hours post infection when replication of positive strand DI RNA was barely observed. Replication of positive strand DI RNAs carrying partial mutations within the 11 nucleotide motif was dependent upon recombination events after transfection. Replication was strongly inhibited when reversion to wild type sequence did not occur, and after recombination, reached similar levels as wild type DI RNA. A DI RNA with mutation upstream of the protein binding motif replicated as efficiently as wild type without undergoing recombination. Thus the conserved 11 nucleotide host protein binding motif appears to play an important role in viral RNA replication. ^

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors

The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-γ-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.