Involvement of the endonuclease domain of the EcoR124I restriction-modification complex in interdomain communication

Type I restriction-modification (R-M) enzymes recognize specific sequences on foreign DNA invading the bacterial cell. At a first sight, also host DNA containing the specific target site for Type I R-M enzyme would cleavage, too, but this doesn't happen as the enzymes are able to distinguish between hostband foreign DNA. Normally the specific sequence on host DNA is either fully methylated or the Type I R-M enzyme recognizes the hemi-methylated state of the DNA, switches to the modification mode and methylates the second strand of the hemi-methylated DNA. Recognition of unmethylated foreign DNA invading the bacterial cell from the outer environment leads to a switching to the restriction mode, initiating endonuclease activity. The R-M complex tightly bound to the recognition sequence on foreign DNA then starts to translocate dsDNA in an ATP-dependent manner towards the stationary enzyme over up to several thousand base pairs. When DNA translocation is finally stalled, the enzyme complex introduces a double strand break, seemingly in a random site distant from the recognition sequence. Multi-subunit structure determines complex behavior of Type I R-M enzymes. The fully assembled Type I R-M enzyme consists of five subunits which are encoded by hsd genes (host specificity for DNA): one copy of HsdS subunit together with two copies of HsdM subunit form the trimeric HsdS1-HsdM2 methyltransferase complex which recognizes and binds to a specific DNA sequence and bears the methylation function. The fully assembled HsdS1-HsdM2-HsdR2 complex possesses ATP-dependent DNA translocation and endonuclease activities located on its HsdR subunits. The X-ray crystal structure of HsdR of EcoR124I with bound ATP gives a first insight of structural/functional correlation in the HsdR subunit. In this work the involvement of the endonuclease domain in interdomain communication within the HsdR motor subunit of EcoR124I is probed experimentally, confronted with computational predictions and discussed in the light of the fully functional pentameric complex

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

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

Dimerisation of the Yeast K⁺ Translocation Protein Trk1 Depends on the K⁺ Concentration

In baker’s yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K+ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K+ concentration

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme

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

Resolved 180s loop.

<p>Electron density (blue mesh) in Lys220Ala mutant HsdR is shown only for the 180s loop, with selected sidechains of the loop shown as sticks in atomic colors with yellow carbons. Outside the 180s loop the sidechains of residues Asp151, Glu165, and Lys167 in the active site, and of Ala220 and Asn221 in the 220s loop, are labeled and shown as sticks, and alpha helix 7 and beta strand f are labeled. The dashed line indicates a distance short enough to permit bonding between the indicated functional groups. Coloring as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.g006" target="_blank">Fig 6</a>.</p

ATP contacts.

<p>Models and electron density are shown for A, WT HsdR; B, Lys220Glu chain A; C, Lys220Arg; D, Lys220Ala. Domain segments (ribbons) and selected residues (stick models) are color-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.g001" target="_blank">Fig 1</a>, with Mg ion shown as a green sphere. Electron density (blue mesh) is shown for ATP (upper center of each panel, atomic colors and orange carbon) and for the 220s loop (lower). The electron density for WT HsdR is better at the same contour level due to its higher resolution, with corresponding differences in the electron density mesh spacing. Dashed lines indicate distances short enough to permit bonding interactions between the indicated functional groups.</p

EcoR124I motor subunit HsdR.

<p>A, Structure of the motor subunit as reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.ref022" target="_blank">22</a>]. The four domains are color-coded: yellow endonuclease, cyan helicase 1, magenta helicase 2 (which together form the translocase); green helical. The structure comprises residues 13–892 of the polypeptide sequence of native HsdR, and is identical with the WT crystal structure 2W00 except for the parts shown in red which were modeled (142–147, 182–189, 585–590, and 859–869) as they are unresolved in the WT crystal structure. ATP is shown as a skeletal model with cyan carbons. Selected side chains relevant for this work are also indicated as skeletal models: Lys220, which contacts ATP, as well as the three conserved catalytic site residues (Asp151, Lys165, and Glu167) are shown in multiple colors with cyan carbons. B, Four domains form a planar array shown as a space-fill cartoon. The four domains are color-coded as in A. Duplex DNA is thought to follow a path down the center of this ‘face’ of the motor subunit, contacting all four domains. Red shading of the endonuclease active site and residue Lys220 that contacts ATP (black skeletal model) emphasizes that the two regions are ~20 Å apart.</p

Crystallographic data collection and refinement statistics.

<p>* Values in parentheses are for the highest-resolution shell.</p><p>Crystallographic data collection and refinement statistics.</p

Cleavage of circular DNA.

<p>Circular plasmid DNA bearing one EcoR124I recognition site was reacted with enzymes reconstituted from HsdS₁HsdM₂ methylase and WT HsdR or mutant HsdRs Lys220Glu, Lys220Ala, or Lys220Arg, and analyzed as in Fig 2. OC, open circular product (▲); L, linear product (●); SC, supercoiled substrate (■); C, control plasmid linearized with HindIII. A: Reactions stopped at the indicated time points were applied to 1.2% agarose gels and visualized by ethidium bromide staining. M is the marker of the indicated numbers of basepairs and C is the linearized plasmind DNA as a control B: Quantification. The three indicated DNA species were quantified individually. Plots for the increase of linear DNA product were derived by fitting an exponential rise to maximum function in SigmaPlot. The points are given for quantification of the gels shown in A, and standard deviations are given from the mean of seven repetitions (WT, Lys220Ala, Lys220Glu) or six repetitions (Lys220Arg) of the experiment conducted with independently purified enzyme preparations.</p

DNA binding.

<p>A. Sequence of synthetic 30-basepair oligonucleotide used, with the EcoR124I recognition sequence shown in bold. B. Electrophoretic mobility-shift assay. The oligonucleotide was 5’ end-labeled with polynucleotide kinase and titrated with EcoR124I reconstituted from HsdS₁HsdM₂ methylase and WT or Lys220Glu HsdR. The oligonucleotide concentration is 5 nM, the concentration of methylase (M2S1 complex) is 40nM, and the concentrations of HsdR are 20, 40, 80, and 120 nM, respectively, in lanes 2–5 (WT) and 8–11 (Lys220Glu). Lane 6, DNA only; lanes 1 and 7, DNA and methylase only. The numbers of subunits in each DNA-protein complex are indicated on the right: R, motor subunit HsdR; M, methylase subunit HsdM; S, specificity subunit HsdS.</p