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. 2016 Sep 21:3:56.
doi: 10.3389/fmolb.2016.00056. eCollection 2016.

Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

José A Ruiz-Masó  1 , Lorena Bordanaba-Ruiseco  1 , Marta Sanz  1 , Margarita Menéndez  2 , Gloria Del Solar  1
Affiliations

Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

José A Ruiz-Masó et al. Front Mol Biosci. .

Abstract

Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed.

Keywords: HUH endonucleases; Mn2+ affinity; RepB thermostability; metal-dependent catalytic activity; plasmid-encoded Rep proteins.

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Figures

Figure 1
Figure 1
RepB6 low-temperature transition involves global changes in the protein structure. (A) Temperature-induced conformational changes of RepB6 as monitored by the variation of the ellipticity at 218 and 282 nm ([Θ] represents protein molar ellipticity). Samples heated at the indicated temperatures (arrows) were analyzed by analytical ultracentrifugation (sedimentation equilibrium), and the estimated average molecular masses are displayed on the protein CD thermal profile. (B) Apparent fraction of modified protein (FDapp) calculated from the transition curves registered at 218 nm (•) and 282 nm (formula image) between 10 and 60°C. The solid line shows the fit of Equation (1) to FDapp values. (C) Temperature transition curves of RepB6 (12 μM) in the presence of increasing concentrations of MnCl2 (indicated inside the graph) measured by CD at 218 nm. The table shows the apparent half-transition temperatures of RepB6 derived from fit of Equation (1) to the figure experimental curves (solid lines). (D) DSC profile of the first thermal transition of RepB6 (30 μM) monitored in the absence and in the presence of 130 μM or 2 mM Mn2+. The position of the maximum of the heat capacity function (Tm) is indicated.
Figure 2
Figure 2
The presence of Mn2+ activates RepB and protects it against thermal inactivation. (A) Activity assays on supercoiled DNA. Samples of RepB unheated, heated to 45°C in the absence of Mn2+, or heated to 45 or 70°C in the presence of 20 mM Mn2+ were mixed, at the indicated molecular ratios, with 6.7 nM of pMV158 supercoiled DNA and then incubated for 30 min at 37°C in the presence of 20 mM MnCl2. The supercoiled (SC), closed relaxed circles (Rel), and open circular (OC) plasmid forms were separated by electrophoresis on agarose gels containing ethidium bromide. Images from different parts of the same gel have been grouped and indicated by dividing lines. (B) RepB6 recognition of the bind DNA measured by EMSA. Unheated or 45°C-heated samples of RepB6 were mixed, at the indicated molecular ratios, with 0.4 μM of a 42-bp DNA fragment containing the three 11 bp-direct repeats that constitute the bind region of the pMV158 dso. Positions of the free (42-bind) and complexed (C1) DNA are indicated. Images from different parts of the same gel have been grouped and indicated by dividing lines. (C) Supercoiled pMV158 DNA (6.7 nM) was incubated, either at 37°C or at 60°C, with the indicated concentrations of Mn2+, in the absence (−) or in the presence (+) of purified RepB6 (RepB:pMV158 DNA molecular ratio of 20:1).
Figure 3
Figure 3
In vitro cleavage activity of RepB6 on ssDNA oligos at different temperatures. A 23-mer oligo substrate (100 nM), radioactively labeled in 5′, was incubated with RepB6 at three different temperatures (30, 37, and 60°C) in the presence of 20 mM of MnCl2 and at the indicated protein:oligo substrate molar ratios. The products were separated on 20% PAA, 8M urea denaturing gels (upper part). Nicking activity of RepB6 was quantified as the percentage of 15-mer product formed (lower part).
Figure 4
Figure 4
Near-UV CD spectrum of RepB (formula image), OBD (formula image), and OD (formula image) separate domains. [Θ] represents the mean residue ellipticity. For comparison, the OBD and OD spectra have been weighted by the fractional contribution of their amino acids to the complete protein sequence.
Figure 5
Figure 5
Temperature-induced changes in the secondary structure of OBD and OBDD42A. Temperature transition curves of RepB6 (•), OBD (formula image), and OBDD42A (formula image) measured by CD at 218 nm in the absence of MnCl2 (A) or in the presence of 0.05 (B), 2 (C), and 5 (D) mM of MnCl2 ([Θ] represents the protein molar ellipticity). The CD thermal profile of RepB6 has been shifted along the ordinate axis to facilitate the comparison with those OBD and OBDD42A. In the case of OBD and OBDD42A, the continuous lines represent an average of the experimental data. Measurements were carried out at 12 μM RepB6 and 19 μM OBD or OBDD42A.
Figure 6
Figure 6
ITC analysis of Mn2+ binding to RepB6, OBD, and OBDD42A. Symbols represent the heat released by mole of Mn2+ injected as a function the Mn2+/protein molar ratio measured at 25°C in ITC buffer. Titrations were performed by adding 1 mM MnCl2 to RepB6(A), OBD (B), or OBDD42A (C) proteins at concentrations ranging from 95 to 119 μM. The binding parameters derived from the fit of the single site binding model to the experimental curve are shown at the bottom and the corresponding theoretical curves are depicted as solid lines.
Figure 7
Figure 7
Nicking and strand-transfer activity of OBD and OBDD42A on ssDNA oligos in the presence of different metal concentrations. (A) Reaction product pattern generated by the nicking and strand-transfer activities of OBD and OBDD42A on ssDNA oligos at the indicated protein:oligo substrate molar ratio and different concentrations of MnCl2. The 27-mer oligo substrate (500 nM), labeled in 3′ with the fluorescent dye Cy5 (indicated by a star), and a 10-fold molar excess of the unlabeled 30-mer oligo were incubated with the protein at 37°C for 1 min. The resultant fluorescent oligos were analyzed by electrophoresis in 20% PAA, 8 M urea denaturing gels and visualized with the aid of FLA-3000 (FUJIFILM) imaging system. A schematic description of the different reaction products is depicted on the right side of the gel image. To compare the reactions products generated by the activity of OBD and OBDD42A the images from different gels acquired and processed under the same conditions have been grouped and indicated by dividing lines. (B) Vertical bar graph comparing the percentage of reaction products rendered by OBD and OBDD42A due to the addition of the indicated concentrations of MnCl2. The assays were performed as depicted in panel A with a protein:oligo substrate molar ratio of 1:10. Vertical bars represent the average value of three different experiments. Errors bars represent standard deviations. The activity increases of OBD and OBDD42A were fitted by nonlinear regression (solid line) to a ligand binding model assuming a single class of binding site for Mn2+. The best fitting values for the apparent dissociation constant for OBD and OBDD42A were 0.5 ± 0.3 and 2.6 ± 0.6 μM, respectively. The percentage of reaction products measured in the absence of MnCl2 added for OBD (formula image) and OBDD42A (formula image) is indicated on the y-axis.
Figure 8
Figure 8
Nicking and strand-transfer activity of RepB6 on ssDNA oligos in the presence of different metal cation concentrations. (A) The vertical bar graph shows the percentage of reaction products rendered by RepB6, in the presence or in the absence of 0.2 mM of MgCl2, when the indicated concentrations of MnCl2 were added. The 27-mer oligo substrate (500 nM), labeled in 3′ with the fluorescent dye Cy5, and a 10-fold molar excess of the unlabeled 30-mer oligo were incubated with the protein at 37°C for 1 min. The assays were performed at a protein:oligo substrate molar ratio of 1:10. Vertical bars represent the average value of three different measurements and error bars are standard deviations. The activity curves of RepB6 supplemented or not with MgCl2 were fitted by nonlinear regression (solid lines) to a ligand binding model assuming a single class of binding site for Mn2+. The best fit values for the apparent dissociation constant for RepB6 was 24.1 ± 6.9 μM in the presence of 0.2 mM of MgCl2, and 80.5 ± 26.2 μM in the absence of MgCl2. The percentage of reaction products measured in the presence of 0.2 mM of MgCl2 with no MnCl2 added (formula image) is indicated on the y-axis. (B) Reaction product pattern generated by the nicking and strand-transfer activities of RepB6 and OBD on ssDNA oligos. The assays were performed as those depicted in (A), at the protein:oligo substrate molar ratio and MnCl2 concentration indicated on the top of each lane. As a control, the reaction was also carried out with NaCl instead of manganese salt. The resultant fluorescent oligos were analyzed, visualized and quantified as in Figure 7. To compare the reactions products generated by the activity of OBD and RepB6 the images from different gels acquired and processed under the same conditions have been grouped and indicated by dividing lines.
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