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Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

Paquet F, Delalande O, Goffinont S, Culard F, Loth K, Asseline U, Castaing B, Landon C - PLoS ONE (2014)

Bottom Line: The polarity of protein binding was determined using paramagnetic probes attached to the DNA.The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry.It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique UPR 4301, Université d'Orléans, Orleans, France.

ABSTRACT
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

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DNA bending ability of the WT and mutant MC1 proteins evaluated by EMSA.EMSA experiment was performed as described for KDapp measurement: 0.1 nM of 5′-[32P]-labeled 26 bp DNA were incubated with the WT or mutant MC1 protein (at 20 nM final concentration, excepting R25A and R25Q mutant versions for which 100 nM was used). At equilibrium, assays were analyzed by EMSA as described in the Materials & Methods section. After 3 hours of electrophoresis, the gel was dried and visualized by autoradiography. The relative electrophoretic mobility of the protein/DNA complex provides an evaluation of the bending ability of the MC1 version considered. With this short DNA duplex, an apparent greater mobility of the nucleoprotein complex is expected for a protein with a greater DNA bending.
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pone-0088809-g005: DNA bending ability of the WT and mutant MC1 proteins evaluated by EMSA.EMSA experiment was performed as described for KDapp measurement: 0.1 nM of 5′-[32P]-labeled 26 bp DNA were incubated with the WT or mutant MC1 protein (at 20 nM final concentration, excepting R25A and R25Q mutant versions for which 100 nM was used). At equilibrium, assays were analyzed by EMSA as described in the Materials & Methods section. After 3 hours of electrophoresis, the gel was dried and visualized by autoradiography. The relative electrophoretic mobility of the protein/DNA complex provides an evaluation of the bending ability of the MC1 version considered. With this short DNA duplex, an apparent greater mobility of the nucleoprotein complex is expected for a protein with a greater DNA bending.

Mentions: Directed mutagenesis was conducted to determine whether amino acids predicted in our models to be in areas of contact with DNA are required for DNA binding. In a previous study, the DNA-binding affinity of Trp61, Trp74 and Met75 mutants was compared with that of the wild-type protein [27]. We showed that the two residues located in the LP5 arm are likely involved in the interaction since substituting Trp74 for Phe and Met75 for Leu led to a decrease in the DNA-binding affinity. On the other hand, and in accordance with our current model, Trp61 located in the β4-strand is probably not directly involved in DNA binding since its substitution for Phe had no effect. In the same study, we also showed that Trp74 and Met75 are involved in DNA bending since their substitution by Ala induced a loss of the capacity of the protein to recognize bent DNA as well as a strong reduction in the protein's ability for DNA bending. Finally, we observed that the substitution Pro76Ala does not affect DNA binding (unpublished results). To complete this analysis, we constructed a new series of MC1 mutants. Altogether, eight proteins were compared for their ability to bind a 26 bp DNA containing the consensus sequence used in the NMR experiments. EMSA were used to separate bound from free DNA (Figure 5 and Supplementary Figure S4) and to determine KD values for each mutant (Table 1). The DNA affinities of the mutants are clearly in accordance with our model of DNA-MC1 binding, implying three area sites. First, we attest that LP5 is involved in DNA binding: substituting Arg71 or Pro72 for Ala induced a slight decrease in the affinity (≈3-fold) whereas the conservative substitution Trp74Phe induced a larger effect (7-fold). A second area involves Ile89 located in the β5-strand, whose substitution for Ala greatly affected the interaction (12.8-fold affinity). Finally, the third point of DNA-MC1 contact was validated by substitutions of Gln23 and Arg25 located in the α-helix. The replacement of Gln23 by a Glu distinctly decreased the binding (7.5-fold affinity), and the effect was larger still with Arg25, whose replacement by Ala or Gln greatly affected the binding (>100-fold affinity). According to the data in the literature, the loss of an electrostatic interaction has a greater effect on DNA binding than the loss of a hydrophobic bond [35]. Mutation of Arg25 strongly affected the binding, which further argues that Arg25 is involved in an electrostatic contact with the DNA backbone.


Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

Paquet F, Delalande O, Goffinont S, Culard F, Loth K, Asseline U, Castaing B, Landon C - PLoS ONE (2014)

DNA bending ability of the WT and mutant MC1 proteins evaluated by EMSA.EMSA experiment was performed as described for KDapp measurement: 0.1 nM of 5′-[32P]-labeled 26 bp DNA were incubated with the WT or mutant MC1 protein (at 20 nM final concentration, excepting R25A and R25Q mutant versions for which 100 nM was used). At equilibrium, assays were analyzed by EMSA as described in the Materials & Methods section. After 3 hours of electrophoresis, the gel was dried and visualized by autoradiography. The relative electrophoretic mobility of the protein/DNA complex provides an evaluation of the bending ability of the MC1 version considered. With this short DNA duplex, an apparent greater mobility of the nucleoprotein complex is expected for a protein with a greater DNA bending.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928310&req=5

pone-0088809-g005: DNA bending ability of the WT and mutant MC1 proteins evaluated by EMSA.EMSA experiment was performed as described for KDapp measurement: 0.1 nM of 5′-[32P]-labeled 26 bp DNA were incubated with the WT or mutant MC1 protein (at 20 nM final concentration, excepting R25A and R25Q mutant versions for which 100 nM was used). At equilibrium, assays were analyzed by EMSA as described in the Materials & Methods section. After 3 hours of electrophoresis, the gel was dried and visualized by autoradiography. The relative electrophoretic mobility of the protein/DNA complex provides an evaluation of the bending ability of the MC1 version considered. With this short DNA duplex, an apparent greater mobility of the nucleoprotein complex is expected for a protein with a greater DNA bending.
Mentions: Directed mutagenesis was conducted to determine whether amino acids predicted in our models to be in areas of contact with DNA are required for DNA binding. In a previous study, the DNA-binding affinity of Trp61, Trp74 and Met75 mutants was compared with that of the wild-type protein [27]. We showed that the two residues located in the LP5 arm are likely involved in the interaction since substituting Trp74 for Phe and Met75 for Leu led to a decrease in the DNA-binding affinity. On the other hand, and in accordance with our current model, Trp61 located in the β4-strand is probably not directly involved in DNA binding since its substitution for Phe had no effect. In the same study, we also showed that Trp74 and Met75 are involved in DNA bending since their substitution by Ala induced a loss of the capacity of the protein to recognize bent DNA as well as a strong reduction in the protein's ability for DNA bending. Finally, we observed that the substitution Pro76Ala does not affect DNA binding (unpublished results). To complete this analysis, we constructed a new series of MC1 mutants. Altogether, eight proteins were compared for their ability to bind a 26 bp DNA containing the consensus sequence used in the NMR experiments. EMSA were used to separate bound from free DNA (Figure 5 and Supplementary Figure S4) and to determine KD values for each mutant (Table 1). The DNA affinities of the mutants are clearly in accordance with our model of DNA-MC1 binding, implying three area sites. First, we attest that LP5 is involved in DNA binding: substituting Arg71 or Pro72 for Ala induced a slight decrease in the affinity (≈3-fold) whereas the conservative substitution Trp74Phe induced a larger effect (7-fold). A second area involves Ile89 located in the β5-strand, whose substitution for Ala greatly affected the interaction (12.8-fold affinity). Finally, the third point of DNA-MC1 contact was validated by substitutions of Gln23 and Arg25 located in the α-helix. The replacement of Gln23 by a Glu distinctly decreased the binding (7.5-fold affinity), and the effect was larger still with Arg25, whose replacement by Ala or Gln greatly affected the binding (>100-fold affinity). According to the data in the literature, the loss of an electrostatic interaction has a greater effect on DNA binding than the loss of a hydrophobic bond [35]. Mutation of Arg25 strongly affected the binding, which further argues that Arg25 is involved in an electrostatic contact with the DNA backbone.

Bottom Line: The polarity of protein binding was determined using paramagnetic probes attached to the DNA.The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry.It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique UPR 4301, Université d'Orléans, Orleans, France.

ABSTRACT
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

Show MeSH
Related in: MedlinePlus