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ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair

Nature Structural & Molecular Biology volume 18, pages 423–431 (2011)Cite this article

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An Erratum to this article was published on 05 March 2012

An Erratum to this article was published on 06 September 2011

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Abstract

The Rad50 ABC–ATPase complex with Mre11 nuclease is essential for dsDNA break repair, telomere maintenance and ataxia telangiectasia–mutated kinase checkpoint signaling. How Rad50 affects Mre11 functions and how ABC–ATPases communicate nucleotide binding and ligand states across long distances and among protein partners are questions that have remained obscure. Here, structures of Mre11–Rad50 complexes define the Mre11 2-helix Rad50 binding domain (RBD) that forms a four-helix interface with Rad50 coiled coils adjoining the ATPase core. Newly identified effector and basic-switch helix motifs extend the ABC–ATPase signature motif to link ATP-driven Rad50 movements to coiled coils binding Mre11, implying an ~30-Å pull on the linker to the nuclease domain. Both RBD and basic-switch mutations cause clastogen sensitivity. Our new results characterize flexible ATP-dependent Mre11 regulation, defects in cancer-linked RBD mutations, conserved superfamily basic switches and motifs effecting ATP-driven conformational change, and they provide a unified comprehension of ABC–ATPase activities.

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Figure 1: The Mre11RBD–Rad50 interface.
Figure 2: A conserved interface links eukaryotic Mre11 and Rad50.
Figure 3: The Mre11–Rad50 interaction interface coordinates DSBR in S. pombe.
Figure 4: The M2R2-head assembly.
Figure 5: Rad50 nucleotide-binding induced conformational changes.
Figure 6: ABC–ATPase superfamily conserved basic-switch residues in Rad50 coordinate DSBR in S. pombe.
Figure 7: Topologically equivalent signature helices connect nucleotide binding to conformational changes in the ABC-ATPase superfamily.

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Change history

  • 21 September 2011

    In the version of this article initially published, panels in Figures 2b (middle row, Dex-LWH), 3b (0.1μM CPT) and 3d (0.1μM CPT) were mistakenly replaced with duplicates of adjacent panels. The errors have been corrected in the HTML and PDF versions of the article.

  • 06 April 2011

    In the version of this article initially published, the acronym ENIGMA was spelled out incorrectly in the Acknowledgments section. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This MRN research is supported by National Cancer Institute grants CA117638 (J.A.T. and P.R.), CA92584 (J.A.T.), CA77325 (P.R.) and in part by the US National Intitutes of Health Intramural Research program 1Z01ES102765-01 (R.S.W.). Microbial complex efforts are supported by the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) Program of the Department of Energy, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 with Lawrence Berkeley National Laboratory (J.A.T.). The Structurally Integrated Biology for Life Sciences (SIBYLS) beamline (BL12.3.1) at the Advanced Light Source is supported by United States Department of Energy program Integrated Diffraction Analysis Technologies DE-AC02-05CH11231 (J.A.T.). We thank G. Hura (Lawrence Berkeley National Laboratory) for expert SAXS data collection assistance.

Author information

Author notes

  1. R Scott Williams, Jessica S Williams & Gabriel Moncalian

    Present address: Present addresses: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA (R.S.W. and J.S.W.) and Instituto de Biomedicina y Biotecnología de Cantabria, Santander, Spain (G.M).,

  2. Gareth J Williams, R Scott Williams, Jessica S Williams and Gabriel Moncalian: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Gareth J Williams, Soumita SilDas & John A Tainer

  2. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

    R Scott Williams, Jessica S Williams, Gabriel Moncalian, Andrew S Arvai, Oliver Limbo, Grant Guenther, Paul Russell & John A Tainer

  3. Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

    R Scott Williams, Gabriel Moncalian, Andrew S Arvai, Grant Guenther & John A Tainer

  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Michal Hammel

  5. Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA

    Paul Russell

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Contributions

G.J.W. analyzed results, did SAXS experiments and wrote the manuscript. J.S.W. and O.L. did S. pombe experiments and analysis. G.M. and R.S.W. solved crystal structures. A.S.A. refined structures. S.S. and G.G. purified proteins. M.H. assisted with SAXS analyses. R.S.W., J.S.W., P.R. and J.A.T. designed research, analyzed results and helped write the manuscript.

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Correspondence to R Scott Williams, Paul Russell or John A Tainer.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 8370 kb)

Supplementary Movie 1

Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate the movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains. (MOV 8250 kb)

Supplementary Movie 2

Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate the movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains (MOV 9395 kb)

Supplementary Movie 3

Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate the movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains (MOV 7497 kb)

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Williams, G., Williams, R., Williams, J. et al. ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair. Nat Struct Mol Biol 18, 423–431 (2011). https://doi.org/10.1038/nsmb.2038

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