NAR Top Articles - Structural Biology
Non-specific protein-DNA interactions control I-CreI target binding and cleavage
Molina, R; Redondo, P; Stella, S; Marenchino, M; D'Abramo, M; Gervasio, FL; Epinat, JC; Valton, J; Grizot, S; Duchateau, P; Prieto, J; Montoya, G
Nucleic Acids Res. (2012) 40 (14): 6936-6945
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Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions...
High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity
Krauss, IR; Merlino, A; Randazzo, A; Novellino, E; Mazzarella, L; Sica, F
Nucleic Acids Res. (2012) 40 (16): 8119-8128
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The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human alpha-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K+ ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of alpha-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na+ or K+ and a circular dichroism study of the structural stability of the aptamer both free and complexed with alpha-thrombin, in the presence of the two ionic species...
Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions
Ohyama, T; Nagata, T; Tsuda, K; Kobayashi, N; Imai, T; Okano, H; Yamazaki, T; Katahira, M
Nucleic Acids Res. (2012) 40 (7): 3218-3231
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Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between beta 1 and alpha 1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date...
The mechanics behind DNA sequence-dependent properties of the nucleosome
Chua, EYD; Vasudevan, D; Davey, GE; Wu, B; Davey, CA
Nucleic Acids Res. (2012) 40 (13): 6338-6352
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Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.
Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods
Bizarro, CV; Alemany, A; Ritort, F
Nucleic Acids Res. (2012) 40 (14): 6922-6935
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RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na+]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg2+ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA...
The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases
Zhang, P; Lee, H; Brunzelle, JS; Couture, JF
Nucleic Acids Res. (2012) 40 (9): 4237-4246
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In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations...
c-MYC promoter G-quadruplex formed at the 5''-end of NHE III1 element: insights into biological relevance and parallel-stranded G-quadruplex stability
Mathad, RI; Hatzakis, E; Dai, JX; Yang, DZ
Nucleic Acids Res. (2011) 39 (20): 9023-9033
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We studied the structures and stabilities of G-quadruplexes formed in Myc1234, the region containing the four consecutive 5' runs of guanines of c-MYC promoter NHE III(1,) which have recently been shown to form in a supercoiled plasmid system in aqueous solution. We determined the NMR solution structure of the 1:2:1 parallel-stranded loop isomer, one of the two major loop isomers formed in Myc1234 in K(+) solution. This major loop isomer, although sharing the same folding structure, appears to be markedly less stable than the major loop isomer formed in the single-stranded c-MYC NHE III(1) oligonucleotide, the Myc2345 G-quadruplex. Our NMR structures indicated that the different thermostabilities of the two 1:2:1 parallel c-MYC G-quadruplexes are likely caused by the different base conformations of the single nucleotide loops. The observation of the formation of the Myc1234 G-quadruplex in the supercoiled plasmid thus points to the potential role of supercoiling in the G-quadruplex formation in promoter sequences. We also performed a systematic thermodynamic analysis of modified c-MYC NHE III(1) sequences...
Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain
Buck, J; Wacker, A; Warkentin, E; Wohnert, J; Wirmer-Bartoschek, J; Schwalbe, H
Nucleic Acids Res. (2011) 39 (22): 9768-9778
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Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR...
Crystal structure of the DNA-bound VapBC2 antitoxin/toxin pair from Rickettsia felis
Mate, MJ; Vincentelli, R; Foos, N; Raoult, D; Cambillau, C; Ortiz-Lombardia, M
Nucleic Acids Res. (2012) 40 (7): 3245-3258
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Besides their commonly attributed role in the maintenance of low-copy number plasmids, toxin/antitoxin (TA) loci, also called 'addiction modules', have been found in chromosomes and associated to a number of biological functions such as: reduction of protein synthesis, gene regulation and retardation of cell growth under nutritional stress. The recent discovery of TA loci in obligatory intracellular species of the Rickettsia genus has prompted new research to establish whether they work as stress response elements or as addiction systems that might be toxic for the host cell. VapBC2 is a TA locus from R. felis, a pathogen responsible for flea-borne spotted fever in humans. The VapC2 toxin is a PIN-domain protein, whereas the antitoxin, VapB2, belongs to the family of swapped-hairpin beta-barrel DNA-binding proteins. We have used a combination of biophysical and structural methods to characterize this new toxin/antitoxin pair. Our results show how VapB2 can block the VapC2 toxin. They provide a first structural description of the interaction between a swapped-hairpin beta-barrel protein and DNA...
Thrombin-aptamer recognition: a revealed ambiguity
Krauss, IR; Merlino, A; Giancola, C; Randazzo, A; Mazzarella, L; Sica, F
Nucleic Acids Res. (2011) 39 (17): 7858-7867
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Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human alpha-thrombin was solved at 2.9-A resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-A resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA...
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