The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that Imi and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright (C) 2004 John Wiley Sons, Ltd.

Evolutionary turnover of mammalian transcription start sites

Alignments of homologous genomic sequences are widely used to identify functional genetic elements and study their evolution. Most studies tacitly equate homology of functional elements with sequence homology. This assumption is violated by the phenomenon of turnover, in which functionally equivalent elements reside at locations that are nonorthologous at the sequence level. Turnover has been demonstrated previously for transcription-factor-binding sites. Here, we show that transcription start sites of equivalent genes do not always reside at equivalent locations in the human and mouse genomes. We also identify two types of partial turnover, illustrating evolutionary pathways that could lead to complete turnover. These findings suggest that the signals encoding transcription start sites are highly flexible and evolvable, and have cautionary implications for the use of sequence-level conservation to detect gene regulatory elements.

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha 1, alpha 2, alpha 1 beta and alpha 2 beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha 2 beta GlyR relative to the alpha 2 GlyR but not in the alpha 1 beta GlyR relative to the alpha 1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha 2 beta GlyR was transferred to the alpha 1 beta GlyR by the G2'A (alpha 1 to alpha 2 subunit) substitution. In addition, the alpha 1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha 1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

PDNAsite:identification of DNA-binding site from protein sequence by incorporating spatial and sequence context

Protein-DNA interactions are involved in many fundamental biological processes essential for cellular function. Most of the existing computational approaches employed only the sequence context of the target residue for its prediction. In the present study, for each target residue, we applied both the spatial context and the sequence context to construct the feature space. Subsequently, Latent Semantic Analysis (LSA) was applied to remove the redundancies in the feature space. Finally, a predictor (PDNAsite) was developed through the integration of the support vector machines (SVM) classifier and ensemble learning. Results on the PDNA-62 and the PDNA-224 datasets demonstrate that features extracted from spatial context provide more information than those from sequence context and the combination of them gives more performance gain. An analysis of the number of binding sites in the spatial context of the target site indicates that the interactions between binding sites next to each other are important for protein-DNA recognition and their binding ability. The comparison between our proposed PDNAsite method and the existing methods indicate that PDNAsite outperforms most of the existing methods and is a useful tool for DNA-binding site identification. A web-server of our predictor (http://hlt.hitsz.edu.cn:8080/PDNAsite/) is made available for free public accessible to the biological research community.

Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Improved Selective Cholesterol Adsorption By Molecularly Imprinted Poly(methacrylic Acid)/silica (pmaa-sio₂) Hybrid Material Synthesized With Different Molar Ratios.

The present paper describes the synthesis of molecularly imprinted polymer - poly(methacrylic acid)/silica and reports its performance feasibility with desired adsorption capacity and selectivity for cholesterol extraction. Two imprinted hybrid materials were synthesized at different methacrylic acid (MAA)/tetraethoxysilane (TEOS) molar ratios (6:1 and 1:5) and characterized by FT-IR, TGA, SEM and textural data. Cholesterol adsorption on hybrid materials took place preferably in apolar solvent medium, especially in chloroform. From the kinetic data, the equilibrium time was reached quickly, being 12 and 20 min for the polymers synthesized at MAA/TEOS molar ratio of 6:1 and 1:5, respectively. The pseudo-second-order model provided the best fit for cholesterol adsorption on polymers, confirming the chemical nature of the adsorption process, while the dual-site Langmuir-Freundlich equation presented the best fit to the experimental data, suggesting the existence of two kinds of adsorption sites on both polymers. The maximum adsorption capacities obtained for the polymers synthesized at MAA/TEOS molar ratios of 6:1 and 1:5 were found to be 214.8 and 166.4 mg g(-1), respectively. The results from isotherm data also indicated higher adsorption capacity for both imprinted polymers regarding to corresponding non-imprinted polymers. Nevertheless, taking into account the retention parameters and selectivity of cholesterol in the presence of structurally analogue compounds (5-α-cholestane and 7-dehydrocholesterol), it was observed that the polymer synthesized at the MAA/TEOS molar ratio of 6:1 was much more selective for cholesterol than the one prepared at the ratio of 1:5, thus suggesting that selective binding sites ascribed to the carboxyl group from MAA play a central role in the imprinting effect created on MIP.

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.