Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping

Abstract EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution.

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Ruthenium(II) Complexes Bearing Thioether-Appended α- Iminopyridine Ligands: Arene Precursors Permit Access to K2-N,N and K3-N,N,S Complexes

10.26434/chemrxiv.9693506.v1 ◽

2019 ◽

Author(s):

Victoria A. Ternes ◽

Hannah A. Morgan ◽

Austin P. Lanquist ◽

Michael P. Murray ◽

Bradley Wile

Keyword(s):

Crystal Structure ◽

Solid State ◽

Strong Field ◽

Active Role ◽

Binding Mode ◽

Phenethyl Alcohol ◽

Ru Complexes

Herein we report the preparation of a series of Ru(II) complexes featuring alpha-iminopyridine ligands bearing thioether functionality (NNSR, where R = Me, CH2Ph, Ph). Metallation using (p cymene)RuCl dimer permits access to (k2-N,N)Ru complexes in which the thioether moiety remains uncoordinated. In the presence of a strong field ligand such as acetonitrile or triphenylphosphine, the p-cymene moiety is displaced, and the ligand adopts a k3-N,N,S binding mode. These complexes are characterized using a combination of solution and solid state methods, including the crystal structure of [(NNSMe)Ru(NCMe)2Cl]Cl. The k2-N,N Ru(II) complexes are shown to serve as efficient precatalysts for the oxidation of sec-phenethyl alcohol at 5 mol% loadings, using a variety of external oxidants and solvents. The complex bearing an S-Ph donor was found to be the most active of those surveyed, suggesting that the thioether donor plays an active role in catalyst speciation for this transformation.

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Faculty Opinions recommendation of Crystal structure of the specificity domain of ribonuclease P.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1012264.194306 ◽

2003 ◽

Author(s):

Douglas Turner

Keyword(s):

Crystal Structure ◽

Ribonuclease P ◽

Specificity Domain

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Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

International Journal of Molecular Sciences ◽

10.3390/ijms22157848 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7848

Author(s):

Annamaria Zannoni ◽

Simone Pelliciari ◽

Francesco Musiani ◽

Federica Chiappori ◽

Davide Roncarati ◽

...

Keyword(s):

Helicobacter Pylori ◽

Response Regulator ◽

Consensus Sequence ◽

Binding Mode ◽

In Vitro Transcription ◽

Target Sequence ◽

Computational Alanine Scanning ◽

Definition Of ◽

Target Promoter

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

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Nucleic acid binding affinity of fd gene 5 protein in the cooperative binding mode.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)43326-6 ◽

1984 ◽

Vol 259 (4) ◽

pp. 2130-2134

Author(s):

A M Bobst ◽

J C Ireland ◽

E V Bobst

Keyword(s):

Nucleic Acid ◽

Binding Affinity ◽

Binding Mode ◽

Cooperative Binding ◽

Nucleic Acid Binding ◽

Gene 5 Protein

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Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)65124-9 ◽

1982 ◽

Vol 257 (11) ◽

pp. 6184-6193

Author(s):

A M Bobst ◽

P W Langemeier ◽

P E Warwick-Koochaki ◽

E V Bobst ◽

J C Ireland

Keyword(s):

Binding Affinity ◽

Bacteriophage T4 ◽

Binding Mode ◽

Cooperative Binding ◽

Gene 32 ◽

T4 Gene 32 Protein

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Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818618116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10763-10772 ◽

Cited By ~ 10

Author(s):

Bernd R. Gardill ◽

Ricardo E. Rivera-Acevedo ◽

Ching-Chieh Tung ◽

Filip Van Petegem

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Binding Mode ◽

Conformational Switch ◽

Channel Inactivation ◽

Dependent Inactivation ◽

Ef Hand ◽

Inherited Arrhythmia ◽

A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

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