Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

• Keywords
• DNA restriction enzymes, Domain interactions, Molecular mechanics, Molecular modeling, Multisubunit enzyme complex, Principal components analysis,
• MeSH
• Adenosine Triphosphate chemistry   MeSH
• Enzyme Activation MeSH
• Principal Component Analysis MeSH
• DNA Helicases chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Protein Interaction Domains and Motifs * MeSH
• Protein Conformation MeSH
• Multienzyme Complexes chemistry   MeSH
• Mutation MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA Helicases MeSH
• Multienzyme Complexes MeSH
• Protein Subunits MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

• Keywords
• DNA restriction enzymes, Domain interactions, E. coli, Molecular modeling, Multisubunit enzyme complex,
• Publication type
• Journal Article MeSH

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

• MeSH
• Adenosine Triphosphate chemistry   metabolism   MeSH
• DNA, Bacterial MeSH
• Escherichia coli genetics   metabolism   MeSH
• Exodeoxyribonuclease V chemistry   genetics   metabolism   MeSH
• Gene Expression MeSH
• Nucleic Acid Conformation MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Plasmids chemistry   metabolism   MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Protein Sorting Signals MeSH
• Escherichia coli Proteins chemistry   genetics   metabolism   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   metabolism   MeSH
• Signal Transduction MeSH
• DNA Cleavage MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA, Bacterial MeSH
• exodeoxyribonuclease V, E coli MeSH Browser
• Exodeoxyribonuclease V MeSH
• HsdR protein, E coli MeSH Browser
• Protein Subunits MeSH
• Protein Sorting Signals MeSH
• Escherichia coli Proteins MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.

• MeSH
• Apoproteins chemistry   isolation & purification   metabolism   MeSH
• Flavin Mononucleotide chemistry   metabolism   MeSH
• Spectrometry, Mass, Electrospray Ionization MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Methionine analogs & derivatives   chemistry   metabolism   MeSH
• Oxidation-Reduction MeSH
• Escherichia coli Proteins chemistry   isolation & purification   metabolism   MeSH
• Repressor Proteins chemistry   isolation & purification   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Apoproteins MeSH
• Flavin Mononucleotide MeSH
• Methionine MeSH
• methionine sulfoxide MeSH Browser
• Escherichia coli Proteins MeSH
• Repressor Proteins MeSH
• WrbA protein, E coli MeSH Browser