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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-binding surface of MC1.The DNA-binding surface of MC1 is represented in green on the solvent-accessible surface. Residues which combine significant CSP and positive charge, or CSP and internal dynamics, are labeled.
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pone-0088809-g001: DNA-binding surface of MC1.The DNA-binding surface of MC1 is represented in green on the solvent-accessible surface. Residues which combine significant CSP and positive charge, or CSP and internal dynamics, are labeled.

Mentions: As attested by the superimposition of 1H-15N HSQC spectra between the free and the DNA-bound form of MC1 (Supplementary Figure S5), the overall structure of the protein is largely unchanged upon binding. Measurement of 1HN and 15N chemical shifts on both the free and bound forms of MC1 yielded the chemical shift perturbations (CSP) along the sequence of MC1. These CSP data were analyzed using the SAMPLEX software [18] and are reported in Supplementary Figure S5. Significantly perturbed residues were located in five sites: the α-Helix from Pro24 to Arg34, three residues in the center of the β3-strand (Ile45-Leu47), six residues belonging to the β4-strand (Phe58-Glu63), part of the loop LP5 (Pro72-Pro76) and practically all of the residues constituting the β5-strand (Val84-Glu90). It is worth noting that chemical shifts can report on both direct interaction and indirect effects such as remote conformational changes [31]–[33]. To refine our selection these CSP data were compared with the electrostatic potential of MC1 and with the flexible regions of MC1 defined by NMR relaxation data [9]. First, the CSP of residues located in the core of MC1 correlate with the basic patch constituted by the positive charges of Arg4, Lys22, Arg25, Lys30, Lys53, Lys54, His56, Arg71, Lys81, Lys85, Lys86, Arg88 and Lys91. Second, we have previously observed that Ile89 and the loop LP5 (67–77), mainly composed of hydrophobic residues (Ala67, Pro68, Pro72, Ala73, Trp74, Met75 and Pro76), possess considerable internal motions on the nanosecond time scale in the free protein and become much less mobile after binding with DNA [9], these residues have significant CSP too. In summary, we assume that residues of MC1, which present CSP after DNA-binding and belong to the basic surface or to the flexible regions, define the DNA-binding surface of MC1 (Figure 1). These residues belong to the α-helix (Arg25, Lys30), the LP5 arm (Pro72, Ala73, Trp74, Met75 and Pro76) and the β5-strand (Lys85, Lys86, Arg88 and Ile89).


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-binding surface of MC1.The DNA-binding surface of MC1 is represented in green on the solvent-accessible surface. Residues which combine significant CSP and positive charge, or CSP and internal dynamics, are labeled.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088809-g001: DNA-binding surface of MC1.The DNA-binding surface of MC1 is represented in green on the solvent-accessible surface. Residues which combine significant CSP and positive charge, or CSP and internal dynamics, are labeled.
Mentions: As attested by the superimposition of 1H-15N HSQC spectra between the free and the DNA-bound form of MC1 (Supplementary Figure S5), the overall structure of the protein is largely unchanged upon binding. Measurement of 1HN and 15N chemical shifts on both the free and bound forms of MC1 yielded the chemical shift perturbations (CSP) along the sequence of MC1. These CSP data were analyzed using the SAMPLEX software [18] and are reported in Supplementary Figure S5. Significantly perturbed residues were located in five sites: the α-Helix from Pro24 to Arg34, three residues in the center of the β3-strand (Ile45-Leu47), six residues belonging to the β4-strand (Phe58-Glu63), part of the loop LP5 (Pro72-Pro76) and practically all of the residues constituting the β5-strand (Val84-Glu90). It is worth noting that chemical shifts can report on both direct interaction and indirect effects such as remote conformational changes [31]–[33]. To refine our selection these CSP data were compared with the electrostatic potential of MC1 and with the flexible regions of MC1 defined by NMR relaxation data [9]. First, the CSP of residues located in the core of MC1 correlate with the basic patch constituted by the positive charges of Arg4, Lys22, Arg25, Lys30, Lys53, Lys54, His56, Arg71, Lys81, Lys85, Lys86, Arg88 and Lys91. Second, we have previously observed that Ile89 and the loop LP5 (67–77), mainly composed of hydrophobic residues (Ala67, Pro68, Pro72, Ala73, Trp74, Met75 and Pro76), possess considerable internal motions on the nanosecond time scale in the free protein and become much less mobile after binding with DNA [9], these residues have significant CSP too. In summary, we assume that residues of MC1, which present CSP after DNA-binding and belong to the basic surface or to the flexible regions, define the DNA-binding surface of MC1 (Figure 1). These residues belong to the α-helix (Arg25, Lys30), the LP5 arm (Pro72, Ala73, Trp74, Met75 and Pro76) and the β5-strand (Lys85, Lys86, Arg88 and Ile89).

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