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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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Model of MC1 interaction with the bent 15 bp DNA.The side chains of K22, P72, W74, M75, K86 and I89 contact the minor groove of DNA and that of R25, with the possible help of Q23, neutralizes the negative phosphates in the shrunk major groove.
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pone-0088809-g004: Model of MC1 interaction with the bent 15 bp DNA.The side chains of K22, P72, W74, M75, K86 and I89 contact the minor groove of DNA and that of R25, with the possible help of Q23, neutralizes the negative phosphates in the shrunk major groove.

Mentions: The average DNA curvature obtained from the 10 models is about 109 ± 6°, which is a much greater angle than that of the starting DNA conformers (1A74: 49.3° and 1YTB: 71.4°) and in agreement with electron microscopy results [13]. The analysis of these models highlights two areas of contact in the minor groove located at the extremities of the 15 bp DNA. The first area is located in the β5-strand around Lys85, Lys86, Arg88 and Ile89 contacting the A-tract. The second area is part of the loop LP5 around Asn70, Arg71, Pro72, Trp74 and Met75 which contacts T16, G17 and G18. Strong additional electrostatic contacts involve A5C6 and the T20G21 phosphate backbone with Arg25 side-chains. These predicted models were in agreement with the experimental DNA-binding surface of MC1 defined above (Figure 1). However, the docking was performed with only one rigid protein model for MC1 and was not representative of the flexibility of the LP5 loop with regard to the core of the protein. We therefore performed a second docking with the commonly used HADDOCK program using the 15 free conformations of MC1 in solution and 10 15 bp DNA models extracted from the previous docking. Both the protein and DNA were considered semi-flexible molecules. Eight models of the DNA-MC1 complex were selected from the more populated clusters (∼140/200 pdb). Their bound DNA is bent with an average curvature of 104±21° (Supplementary Figure S9). The angle of curvature depends on the position of the loop LP5 with regard to the core of MC1. As in the first models, the protein hangs on to the extremities of the DNA by inserting the expected residues Pro72, Trp74, Met75, Lys85, Lys86, Arg88 and Ile89 in the minor groove. The side chain of Lys22 is most probably involved in the minor groove interaction as well (Figure 4). As a consequence of this dramatic curvature, the A5pC6 and T20pG21 phosphates move closer to each other and the side chains of Arg25 and Gln23, which were not constrained by our ambiguous interaction restraints (AIRs) during the docking, are positioned in the major groove (Figure 4). In the center of the 15 bp oligonucleotide (CACACA region) the minor groove is widened and shallow whereas the major groove is narrow and deep (Supplementary Figure S9).


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)

Model of MC1 interaction with the bent 15 bp DNA.The side chains of K22, P72, W74, M75, K86 and I89 contact the minor groove of DNA and that of R25, with the possible help of Q23, neutralizes the negative phosphates in the shrunk major groove.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088809-g004: Model of MC1 interaction with the bent 15 bp DNA.The side chains of K22, P72, W74, M75, K86 and I89 contact the minor groove of DNA and that of R25, with the possible help of Q23, neutralizes the negative phosphates in the shrunk major groove.
Mentions: The average DNA curvature obtained from the 10 models is about 109 ± 6°, which is a much greater angle than that of the starting DNA conformers (1A74: 49.3° and 1YTB: 71.4°) and in agreement with electron microscopy results [13]. The analysis of these models highlights two areas of contact in the minor groove located at the extremities of the 15 bp DNA. The first area is located in the β5-strand around Lys85, Lys86, Arg88 and Ile89 contacting the A-tract. The second area is part of the loop LP5 around Asn70, Arg71, Pro72, Trp74 and Met75 which contacts T16, G17 and G18. Strong additional electrostatic contacts involve A5C6 and the T20G21 phosphate backbone with Arg25 side-chains. These predicted models were in agreement with the experimental DNA-binding surface of MC1 defined above (Figure 1). However, the docking was performed with only one rigid protein model for MC1 and was not representative of the flexibility of the LP5 loop with regard to the core of the protein. We therefore performed a second docking with the commonly used HADDOCK program using the 15 free conformations of MC1 in solution and 10 15 bp DNA models extracted from the previous docking. Both the protein and DNA were considered semi-flexible molecules. Eight models of the DNA-MC1 complex were selected from the more populated clusters (∼140/200 pdb). Their bound DNA is bent with an average curvature of 104±21° (Supplementary Figure S9). The angle of curvature depends on the position of the loop LP5 with regard to the core of MC1. As in the first models, the protein hangs on to the extremities of the DNA by inserting the expected residues Pro72, Trp74, Met75, Lys85, Lys86, Arg88 and Ile89 in the minor groove. The side chain of Lys22 is most probably involved in the minor groove interaction as well (Figure 4). As a consequence of this dramatic curvature, the A5pC6 and T20pG21 phosphates move closer to each other and the side chains of Arg25 and Gln23, which were not constrained by our ambiguous interaction restraints (AIRs) during the docking, are positioned in the major groove (Figure 4). In the center of the 15 bp oligonucleotide (CACACA region) the minor groove is widened and shallow whereas the major groove is narrow and deep (Supplementary Figure S9).

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