共查询到20条相似文献,搜索用时 31 毫秒
1.
Fujikawa N Kurumizaka H Nureki O Terada T Shirouzu M Katayama T Yokoyama S 《Nucleic acids research》2003,31(8):2077-2086
Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 Å resolution. DnaA domain IV contains a helix–turn–helix motif for DNA binding. One helix and a loop of the helix– turn–helix motif are inserted into the major groove and 5 bp (3′ two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5′ one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by ~28° was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin. 相似文献
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Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information
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In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix–turn–helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of ‘partial’ domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function. 相似文献
4.
Identifying DNA-binding proteins using structural motifs and the electrostatic potential 总被引:3,自引:0,他引:3
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Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. 相似文献
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Using structural motif templates to identify proteins with DNA binding function 总被引:3,自引:2,他引:1
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This work describes a method for predicting DNA binding function from structure using 3-dimensional templates. Proteins that bind DNA using small contiguous helix–turn–helix (HTH) motifs comprise a significant number of all DNA-binding proteins. A structural template library of seven HTH motifs has been created from non-homologous DNA-binding proteins in the Protein Data Bank. The templates were used to scan complete protein structures using an algorithm that calculated the root mean squared deviation (rmsd) for the optimal superposition of each template on each structure, based on Cα backbone coordinates. Distributions of rmsd values for known HTH-containing proteins (true hits) and non-HTH proteins (false hits) were calculated. A threshold value of 1.6 Å rmsd was selected that gave a true hit rate of 88.4% and a false positive rate of 0.7%. The false positive rate was further reduced to 0.5% by introducing an accessible surface area threshold value of 990 Å2 per HTH motif. The template library and the validated thresholds were used to make predictions for target proteins from a structural genomics project. 相似文献
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Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain. CcO catalyzes a four electron reduction of O2 to water at a catalytic site formed by high-spin heme (a3) and copper atoms (CuB). While it is recognized that proton movement is coupled to oxygen reduction, the proton channel(s) have not been well defined. Using computational methods developed to study protein topology, membrane channels and 3D packing arrangements within transmembrane (TM) helix arrays, we find that subunit-1 (COX-1), subunit-2 (COX-2) and subunit-3 (COX-3) contribute 139, 46 and 25 residues, respectively, to channel formation between the mitochondrial matrix and intermembrane space. Nine of 12 TM helices in COX-1, both helices in COX-2 and 5 of the 6 TM helices in COX-3 are pore-lining regions (possible channel formers). Heme a3 and the CuB sites (as well as the CuA center of COX-2) are located within the channel that includes TM-6, TM-7, TM-10 and TM-11 of COX-1 and are associated with multiple cholesterol and caveolin-binding (CB) motifs. Sequence analysis identifies five CB motifs within COX-1, two within COX-2 and four within COX-3; each caveolin containing a pore-lining helix C-terminal to a TM helix–turn–helix. Channel formation involves interaction between multiple pore-lining regions within protein subunits and/or dimers. PoreWalker analysis lends support to the D-channel model of proton translocation. Under physiological conditions, caveolins may introduce channel formers juxtaposed to those in COX-1, COX-2 and COX-3, which together with cholesterol may form channel(s) essential for proton translocation through the inner mitochondrial membrane. 相似文献
9.
Carol H. Pong Christopher J. Harmer Janine K. Flores Sandro F. Ataide Ruth M. Hall 《The Journal of biological chemistry》2021,297(4)
The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family. 相似文献
10.
Verdemato PE Brannigan JA Damblon C Zuccotto F Moody PC Lian LY 《Nucleic acids research》2000,28(19):3710-3718
The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151–160) which form the recognition helix and the ‘wing’ of a helix–turn–wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O6-methylguanine (O6meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain. 相似文献
11.
Siu Wah Wong-Deyrup Youngbae Kim Sonya J. Franklin 《Journal of biological inorganic chemistry》2006,11(1):17-25
The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis
mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed
based on the structural similarity of the helix–turn–helix motif of homeodomains and the EF-hand motifs of calmodulin, as
previously described for P3W. Like P3W, P5b binds both Eu(III) (K
d=12.6±1.9 μM) and Ca(II) (K
d=70±8 μM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified
one target family for CaP5b [5′-pur-T-pur-G-(G/C)-3′], and two target sites for CaP3W [5′-(A/T)-G-G-G-(T/C)-3′ and 5′-A-T-(G/T)-T-G-3′].
Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA
containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly.
These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides
have been found to bind selectively to DNA targets, analogous to natural protein–DNA interactions. This corroborates our earlier
conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
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The transactivator Staf, which contains seven contiguous zinc fingers of the C2-H2 type, exerts its effects on gene expression by binding to specific targets in vertebrate small nuclear RNA (snRNA) and snRNA-type gene promoters. Here, we have investigated the interaction of the Staf zinc finger domain with the optimal Xenopus selenocysteine tRNA (xtRNASec) and human U6 snRNA (hU6) Staf motifs. Generation of a series of polypeptides containing increasing numbers of Staf zinc fingers tested in binding assays, by interference techniques and by binding site selection served to elucidate the mode of interaction between the zinc fingers and the Staf motifs. Our results provide strong evidence that zinc fingers 3–6 represent the minimal zinc finger region for high affinity binding to Staf motifs. Furthermore, we show that the binding of Staf is achieved through a broad spectrum of close contacts between zinc fingers 1–6 and xtRNASec or optimal sites or between zinc fingers 3–6 and the hU6 site. Extensive DNA major groove contacts contribute to the interaction with Staf that associates more closely with the non-template than with the template strand. Based on these findings and the structural information provided by the solved structures of other zinc finger–DNA complexes, we propose a model for the interaction between Staf zinc fingers and the xtRNASec, optimal and hU6 sites. 相似文献
14.
Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix–turn–helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix α1 contacts tRNA whereas helix α2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import. 相似文献
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Wong-Deyrup SW Prasannan C Dupureur CM Franklin SJ 《Journal of biological inorganic chemistry》2012,17(3):387-398
Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously
reported the design and characterization of an engineered “metallohomeodomain” protein (C2) based on a chimera of the EF-hand
Ca-binding motif and the helix–turn–helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159–2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference
and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function
by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence
spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments,
metallated F2 exhibits a specificity toward 5′-TAATTA-3′ sequences. Finally, Ca2F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our
knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step
forward in the design of artificial metallonucleases by utilizing biologically significant metal ions. 相似文献
17.
The poly-proline type II extended left-handed helical structure is well represented in proteins. In an effort to determine the helix's role in nucleic acid recognition and binding, a survey of 258 nucleic acid-binding protein structures from the Protein Data Bank was conducted. Results indicate that left-handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA-2, the Epstein-Barr nuclear antigen-1, and the Drosophila paired protein homeodomain all contain left-handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left-handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being "unstructured." Proposed mechanisms for nucleic acid interactions by the extended left-handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main-chain and side-chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar-phosphates, respectively. Our results emphasize the need to classify the left-handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix-turn-helix, zinc fingers, leucine zippers, and others. 相似文献
18.
Junichi Taira Mitsukuni Shibue Satoshi Osada Hiroaki Kodama 《International journal of peptide research and therapeutics》2010,16(4):277-282
Characteristic motifs have been identified in natural channel forming peptides though critical roles of such motifs are not
well understood. In this paper, the helix‐kink motif found in peptaibols was embedded into the α-aminoisobutyric acid (Aib)
rich template to explore its roles in peptide structure and ion channel functions. According to circular dichroism studies
and single channel measurements, the motif reduced helical contents of peptide whereas ion channel forming was facilitated
and conductance value was increased. 相似文献
19.
Zinc finger peptides for the regulation of gene expression. 总被引:11,自引:0,他引:11
A Klug 《Journal of molecular biology》1999,293(2):215-218
20.
Metal ions are crucial for nucleic acid folding. From the free energy landscapes, we investigate the detailed mechanism for ion-induced collapse for a paradigm system: loop-tethered short DNA helices. We find that Na+ and Mg2+ play distinctive roles in helix–helix assembly. High [Na+] (>0.3 M) causes a reduced helix–helix electrostatic repulsion and a subsequent disordered packing of helices. In contrast, Mg2+ of concentration >1 mM is predicted to induce helix–helix attraction and results in a more compact and ordered helix–helix packing. Mg2+ is much more efficient in causing nucleic acid compaction. In addition, the free energy landscape shows that the tethering loops between the helices also play a significant role. A flexible loop, such as a neutral loop or a polynucleotide loop in high salt concentration, enhances the close approach of the helices in order to gain the loop entropy. On the other hand, a rigid loop, such as a polynucleotide loop in low salt concentration, tends to de-compact the helices. Therefore, a polynucleotide loop significantly enhances the sharpness of the ion-induced compaction transition. Moreover, we find that a larger number of helices in the system or a smaller radius of the divalent ions can cause a more abrupt compaction transition and a more compact state at high ion concentration, and the ion size effect becomes more pronounced as the number of helices is increased. 相似文献