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Conselho Nacional de Desenvolvimento Científico e Tecnológico ASR Proteins are ABA, stress and ripening induced proteins present in various plant species such as lily (Lilium longiflorum), sugarcane (Saccharum officinarum), grape (Vitis vinifera) ; Rice (Oryza sativa), petunia, banana (Musa acuminata and Musa balbisiana) and corn (Zea mays). However no single ASR gene has been found in Arabidopsis. ASRs are small, hydrophilic and unstructured proteins. Its function, as well as the signaling pathway to which they belong, remains unknown. In soybean were reported the presence of three ASRs, which were called GmASR1, and GmASR2 GmASR3. This study aimed to identify factors that interact with the GmASR3 promoter region, as well as factors that bind to the protein GmASR3. After analysis of databases, the promoter region about 500 bp upstream of the transcription initiation codon was obtained from PCR performed with genomic DNA from soybean (Glycine max), and cloned into plasmid pHIS2.1. This construct was transformed in yeast S. cerevisiae W303 for subsequent liquid screening of cDNAs that express factors capable of interacting with the promoter GmASR3 by mono-hybrid yeast system. It was found that the protein GmASR3 has weak transactivation capacity under the used conditions, which enabled the realization of two-hybrid experiments to identify factors that bind to the protein GmASR3.The factors that bind to GmASR3 were obtained by two-hybrid system yeast, using for this yeast S. cerevisiae AH109 carrying the plasmid pDest32 which was inserted the cDNA sequence GmASR3. Likewise, the method of liquid screening in deep well plates was used. The colonies that were considered to have the factors that interact with the promoter or the protein in GmASR were re-transformed in E. coli Dh5α and sent for sequencing. Proteínas ASR (ABA, Stress and ripening induced protein) são induzidas em situações de estresses, maturação ou na presença de ABA, em diversas espécies vegetais, como Lírio (Lilium longiflorum); cana de açúcar (Saccharum officinarum); uva (Vitis vinífera); arroz (Oriza sativa); petúnia, banana (Musa acuminata e Musa balbisiana) e milho (Zea mays), entre outros. No entanto nenhum gene ASR foi encontrado em Arabidopsis. ASRs são proteínas pequenas, hidrofílicas e sem estrutura definida. Sua função é desconhecida, assim como a via de sinalização a qual pertencem. Em soja foi relatada a presença de três ASRs, que foram denominadas GmASR1, GmASR2 e GmASR3. Este trabalho buscou identificar proteínas que interagem com a região promotora de GmASR3, assim como fatores que se ligam à proteína GmASR3. Após analise em bancos de dados, a região promotora em torno de 500pb a montante do códon de iniciação da transcrição foi obtida a partir de PCR realizado com o DNA genômico de soja (Glycine max, cultivar CD206) e clonado em plasmídeo pHIS2.1. Esta construção foi transformada em leveduras S. cerevisiae W303 para posterior triagem líquida de cDNAs que expressam fatores capazes de interagir com o promotor de GmASR3, a partir do método do mono-híbrido de leveduras. Foi verificado que a proteína GmASR3 possui fraca capacidade de transativação nas condições utilizadas, o que possibilitou a realização de experimentos de duplo-híbrido com o fim de identificar fatores que se ligam à proteína GmASR3. Os fatores que se ligam a GmASR3 foram obtidos através do método do duplo-híbrido de leveduras, utilizando para isso leveduras S. cerevisiae AH109 portadoras do plasmídeo pDest32 ao qual foi inserido a sequencia do cDNA de GmASR3. As triagens de mono e duplo-híbrido foram realizadas utilizando o método da triagem líquida em placas deep well. As colônias que foram consideradas portadoras dos fatores que interagem com o promotor ou com a proteína GmASR foram retransformados em E. coli Dh5α e enviados para sequenciamento.

Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein’s function is not trivial. Protein design tools are commonly used to explain mutations that affect protein stability, or protein–protein interaction, but not for mutations that could affect protein–DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several diseases such as aniridia. The validity of FoldX to deal with protein–DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos. We conclude that 88% of the Pax6 mutations can be linked to changes in intrinsic stability (77%) and/or to its capabilities to bind DNA (30%). Our study emphasizes the importance of structure-based analysis to understand the molecular basis of diseases and shows that protein–DNA interactions can be analyzed to the same level of accuracy as protein stability, or protein–protein interactions., National Institutes of Health (U.S.) (R01 HG003985), National Human Genome Research Institute (U.S.) (R01 HG003985)

Nucleic acid recognition is often mediated by alpha-helices or disordered regions that fold into alpha-helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical alpha-helix is stabilized at the N-terminus, while the PII forms at the C-terminus half of the peptide. Re-examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N-terminus half is a canonical alpha-helix, while the C-terminal half adopts a 3(10) helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10(4)-fold lower affinity, and 500-fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained alpha-helix conformation, "presented" by this unique architecture. Fil: Wetzler, Diana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Gallo, Mariana. Universita Tor Vergata; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Melis, Riccardo. Universita Tor Vergata; Italia Fil: Eliseo, Tomasso. Universita Tor Vergata; Italia Fil: Nadra, Alejandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universita Tor Vergata; Italia. Centro de Regulación Genómica; España Fil: Ferreiro, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Paci, Maurizio. Universita Tor Vergata; Italia Fil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Cicero, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universita Tor Vergata; Italia Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

The Epstein-Barr nuclear antigen 1 (EBNA1) is essential for DNA replication and episome segregation of the viral genome, and participates in other gene regulatory processes of the Epstein-Barr virus in benign and malignant diseases related to this virus. Despite the participation of other regions of the protein in evading immune response, its DNA binding, dimeric beta-barrel domain (residues 452-641) is necessary and sufficient for the main functions. This domain has an unusual topology only shared by another viral origin binding protein (OBP), the E2 DNA binding domain of papillomaviruses. Both the amino acid and DNA target sequences are completely different for these two proteins, indicating a link between fold conservation and function. In this work we investigated the folding and stability of the DNA binding domain of EBNA1 OBP and found it is extremely resistant to chemical, temperature, and pH denaturation. The thiocyanate salt of guanidine is required for obtaining a complete transition to a monomeric unfolded state. The unfolding reaction is extremely slow and shows a marked uncoupling between tertiary and secondary structure, indicating the presence of intermediate species. The Gdm.SCN unfolded protein refolds to fully soluble and spherical oligomeric species of 1.2 MDa molecular weight, with identical fluorescence centre of spectral mass but different intensity and different secondary structure. The refolded spherical oligomers are substantially less stable than the native recombinant dimer. In keeping with the substantial structural rearrangement in the oligomers, the spherical oligomers do not bind DNA, indicating that the DNA binding site is either disrupted or participates in the oligomerization interface. The puzzling extreme stability of a dimeric DNA binding domain from a protein from a human infecting virus in addition to a remarkable kinetically driven folding where all molecules do not return to the most stable original species suggests a co-translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed Fil: Freire Espeleta, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Oddo, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Frappier, Lory. University of Toronto; Canadá Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Despite decades of investigations, it is not yet clear whether there are rules dictating the specificity of the interaction between amino acids and nucleotide bases. This issue was addressed by determining, in a dataset consisting of 100 high-resolution protein-DNA structures, the frequency and energy of interaction between each amino acid and base, and the energetics of water-mediated interactions. The analysis was carried out using HINT, a non-Newtonian force field encoding both enthalpic and entropic contributions, and Rank, a geometry-based tool for evaluating hydrogen bond interactions. A frequency- and energy-based preferential interaction of Arg and Lys with G, Asp and Glu with C, and Asn and Gln with A was found. Not only favorable, but also unfavorable contacts were found to be conserved. Water-mediated interactions strongly increase the probability of Thr-A, Lys-A, and Lys-C contacts. The frequency, interaction energy, and water enhancement factors associated with each amino acid–base pair were used to predict the base triplet recognized by the helix motif in 45 zinc fingers, which represents an ideal case study for the analysis of one-to-one amino acid–base pair contacts. The model correctly predicted 70.4% of 135 amino acid–base pairs, and, by weighting the energetic relevance of each amino acid–base pair to the overall recognition energy, it yielded a prediction rate of 89.7%. © 2008 Wiley Periodicals, Inc. J Comput Chem 2008

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Recognition between (bio)(macro)molecules is crucial for all aspects of life. Although the molecular pathways can be complicated and network structured many of the recognition phenomena are based on single molecule interactions. Due to exponentially expanding number of known 3D (three dimensional) structures of (for biomedical research) target macromolecules it is important to develop accurate and efficient computer methods that will use the full information contained therein. We developed a method based on molecular modelling and 3D QSAR (Quantitative Structure Activity Relationship). Outline of the method and results obtained for two different systems will be presented. a) Lipase Study was performed in order to derive a model for enantioselectivity of bacterial lipase, namely lipase from of Burkholderia Cepacia (BCL), towards primary and secondary alcohols. The most probable conformers of the substrate tetrahedral intermediates were elucidated and their interactions with the amino acid residues at the binding pocket(s) were calculated. The free energy difference between TIs of two enantiomers was approximated by the residue-based sum of potential energy differences and the solvent accessible surface area differences. The results obtained can be used to predict enzyme enantioselectivity in a substrate transformation as well as to plan point mutations of the enzyme to achieve high enantioselectivity in the enzyme catalysed transformation of specific substrates. b) Nuclear receptor DNA binding To identify the major determinants of the DNA binding specificity of nuclear transcription factors, two sets of glucocorticoid receptor transcription factor mutants binding to different panlindromic elements were studied. It was found that the parameters important for binding, non-specific and specific, of nuclear transcription factors to DNA are similar in both sets of complexes. The dominating parameters are solvation free energy of the central (mutated) bases of a hexameric half site and the electrostatic interaction between the central nucleotides and the mutated residues (residues from so called P-box) as well as some more distant charged amino acid residues.

Protein recognition of specific DNA binding sites is critical for cellular processes; transcription, replication, and restriction are but a few of the biological activities regulated by DNA binding proteins. Although DNA can exist in any number of sequences and conformations, proteins have the exquisite capability of recognizing specific nucleotide sequences in an entire genome. Chapter One is a review that concentrates on sequence-specific DNA binding proteins primarily capable of regulating gene expression and also able to serve structural and catalytic roles in biological processes. The helix-turn-helix repressor proteins from phages λ and 434 and lac and trp repressors, leucine zipper proteins, zinc-finger proteins, the homeodomain, basic helixloop-helix proteins, arc and mnt repressors, and double helix-turn-helix proteins are discussed. Affinity cleaving studies on mutants of the DNA binding domain of Hin recombinase, Hin(139-190), are discussed in Chapter Two. Arg140 has been previously found to be extremely important for sequence-specific binding of Hin(139-190) to the hixL operator. Mutants in which Arg140 of Hin(139-190) is replaced with Lys, Ala, β-Ala, Glu, Gly, and GIn were studied; a mutant in which Arg142 is replaced by Lys is also discussed. The binding affinities and sequence specificities of these mutants are very different from that of Hin(139- 190). Also Hin(139-184), in which the six carboxyl terminal residues are removed, was studied by affinity cleaving and shown to have a similar sequence specificity, albeit reduced binding affinity, compared to Hin(139-190). In Chapter Three, a procedure for quantitating thermodynamic parameters using the affinity cleaving technique is discussed. In this procedure, affinity cleaving reactions are run over a wide range of protein concentrations. A binding isotherm is generated and fit, using a least-squares program, and reproducible values for binding constants can be obtained. Binding constants were quantitated for Hin(39-190), two of the mutant proteins, and Hin(139-184). This methodology was developed in order to obtain thermodynamic parameters of complexation between these synthesized proteins and their DNA binding sites. Binding constants were measured only for the hixL IRL and IRR sites for each protein, although in some cases, strong binding was also exhibited at the secondary and even tertiary sites. This method can resolve binding curves for individual sites in a cooperative system. Typical binding constants ranged between 105 M-1 and 107 M-1 at 20mM NaCI for the hixL site. Affinity cleaving studies were performed on the DNA binding domain of lac repressor in Chapter Four. Based upon sequence homology analysis between the binding domains of lac and cro repressors, the DNA binding interaction between the lac repressor and operator was assigned as a helix-turn-helix motif. NMR studies on the DNA binding domain of lac repressor have shown that the recognition helix of lac binds the major groove of DNA in an orientation opposite that of cro. Affinity cleaving studies on the lac repressor DNA binding domain support the NMR results and establish the orientation of the recognition helix of the lac repressor. In Chapter Five are discussed affinity cleaving studies on the engrailed homeodomain, for which a high-resolution cocrystal structure was recently published. Until now, affinity cleaving has been performed on proteins for which no high-resolution structural data exist. Because the engrailed homeodomain contains sequence elements very similar to Hin(139-190), it was chosen for affinity cleaving studies in order to compare and to interpret structural data obtained by affinity cleaving with a protein of known structure.

When it comes to protein specificity and function their three-dimensional structure is the ultimate determinant. Thus, sequences that participate in key parts, such as catalytic sites or DNA binding have been favored and maintained highly conserved during evolution. However, in a reversal of fortune, selection has favored conservation of shapes over sequence, especially when proteins look like nucleic acids. Proteins from pathogens evade the host's defenses because they are shaped as DNA; others use such a disguise for transcriptional regulation. Several factors are tRNA look-alikes so that they can efficiently control the process of protein synthesis. Molecular mimicry among RNAs could result in a new unexplored level in gene regulation. This comprehensive review outlines this important area and aims to emphasize that molecular mimicry could in fact be more widespread than initially thought and eventually adds a new layer of genetic regulation.

Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo.

Journal of Biomolecular NMR, 69 (3), ISSN:0925-2738, ISSN:1573-5001

Cis2-His2 zinc finger (C2H2-ZF) proteins are the largest family of transcription factors in human and higher metazoans. However, the DNA-binding preferences of many members of this family remain unknown. We have developed a computational method to predict these DNA-binding preferences. We combine information from crystal structures composed by C2H2-ZF domains and from bacterial one-hybrid experiments to compute scores for protein-DNA binding based on statistical potentials. We apply the scores to compute theoretical position weight matrices (PWMs) of proteins with a DNA-binding domain composed by C2H2-ZF domains, with the only requirement of an input structure (experimentally determined or modelled). We have tested the capacity to predict PWMs of zinc finger domains, successfully predicting 3-2 nucleotides of a trinucleotide binding site for about 70% variants of single zinc-finger domains of Zif268. We have also tested the capacity to predict the PWMs of proteins composed by three C2H2-ZF domains, successfully matching between 60% and 90% of the binding-site motif according to the JASPAR database. The tests are used as a proof of the capacity to scan a DNA fragment and find the potential binding sites of transcription-factors formed by C2H2-ZF domains. As an example, we have tested the approach to predict the DNA-binding preferences of the human chromatin binding factor CTCF.