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NAR Top Articles - Structural Biology

Structural Biology

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December 2013


5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases
Le Bihan, YV; Izquierdo, MA; Coste, F; Aller, P; Culard, F; Gehrke, TH; Essalhi, K; Carell, T; Castaing, B
Nucleic Acids Res. (2011) 39 (14): 6277-6290
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DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.

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...

Novel dimeric structure of phage {phi}29-encoded protein p56: insights into uracil-DNA glycosylase inhibition
Asensio, JL; Perez-Lago, L; Lazaro, JM; Gonzalez, C; Serrano-Heras, G; Salas, M
Nucleic Acids Res. (2011) 39 (22): 9779-9788
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Protein p56 encoded by the Bacillus subtilis phage phi 29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel beta-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG...

The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1
Lim, SC; Bowler, MW; Lai, TF; Song, HW
Nucleic Acids Res. (2012) 40 (21): 11009-11022
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Mutations in immunoglobulin m-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30 degrees and 10 degrees, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.

Recognition of methylated DNA through methyl-CpG binding domain proteins
Zou, XQ; Ma, W; Solov'yov, IA; Chipot, C; Schulten, K
Nucleic Acids Res. (2012) 40 (6): 2747-2758
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DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines through an interplay of hydrogen bonding and cation-pi interaction. Through molecular dynamics and quantum-chemistry calculations we investigate the methyl-cytosine recognition process and demonstrate that methylation enhances MBD-mDNA binding by increasing the hydrophobic interfacial area and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex.

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...

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...

The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex
Petit, AP; Wohlbold, L; Bawankar, P; Huntzinger, E; Schmidt, S; Izaurralde, E; Weichenrieder, O
Nucleic Acids Res. (2012) 40 (21): 11058-11072
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The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to decay. The conserved core of the complex consists of a catalytic module comprising two deadenylases (CAF1/POP2 and CCR4a/b) and the NOT module, which contains at least NOT1, NOT2 and NOT3. NOT1 bridges the interaction between the two modules and therefore, acts as a scaffold protein for the assembly of the complex. Here, we present the crystal structures of the CAF1-binding domain of human NOT1 alone and in complex with CAF1. The NOT1 domain comprises five helical hairpins that adopt an MIF4G (middle portion of eIF4G) fold. This NOT1 MIF4G domain binds CAF1 through a pre-formed interface and leaves the CAF1 catalytic site fully accessible to RNA substrates. The conservation of critical structural and interface residues suggests that the NOT1 MIF4G domain adopts a similar fold and interacts with CAF1 in a similar manner in all eukaryotes...

Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb2+
Liu, W; Zhu, H; Zheng, B; Cheng, S; Fu, Y; Li, W; Lau, TC; Liang, HJ
Nucleic Acids Res. (2012) 40 (9): 4229-4236
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DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb2+-induced G-quadruplex formed unimolecularly and the highest melting temperature (T-m) is 72 degrees C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb2+ ions to PS2.M is two, suggesting that the Pb2+ ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb2+-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO3)(2) to the Na+- or K+-induced G-quadruplexes, which may be attributed to the replacement of Na+ or K+ by Pb2+ ions...

Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers
Baburajendran, N; Jauch, R; Tan, CYZ; Narasimhan, K; Kolatkar, PR
Nucleic Acids Res. (2011) 39 (18): 8213-8222
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Smad proteins form multimeric complexes consisting of the 'common partner' Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-beta and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein-protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored...

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