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Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

Andrew Lyon, Alex Zelter, Shruthi Viswanath, Alison Maxwell, Richard Johnson, King Clyde B. Yabut, Michael MacCoss, Trisha N. Davis, Eric Muller, Andrej Sali, David A. Agard
doi: https://doi.org/10.1101/311027
Andrew Lyon
1Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143
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Alex Zelter
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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Shruthi Viswanath
3Department of Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, CA 94143
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Alison Maxwell
1Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143
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Richard Johnson
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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King Clyde B. Yabut
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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Michael MacCoss
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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Trisha N. Davis
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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Eric Muller
2Department of Biochemistry, University of Washington, Seattle, WA 98195
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Andrej Sali
3Department of Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, CA 94143
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David A. Agard
1Department of Biochemistry and Biophysics and the Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143
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Abstract

Microtubule (MT) nucleation in vivo is regulated by the γ-tubulin ring complex (γTuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast γTuRC is composed of seven γ-tubulin small complex (γTuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple γTuSCs, thereby stabilizing the otherwise low-affinity interface between γTuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with γTuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the γTuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and γTuSC and the other stabilizing the interface between adjacent γTuSCs.

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Posted April 30, 2018.
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Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly
Andrew Lyon, Alex Zelter, Shruthi Viswanath, Alison Maxwell, Richard Johnson, King Clyde B. Yabut, Michael MacCoss, Trisha N. Davis, Eric Muller, Andrej Sali, David A. Agard
bioRxiv 311027; doi: https://doi.org/10.1101/311027
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Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly
Andrew Lyon, Alex Zelter, Shruthi Viswanath, Alison Maxwell, Richard Johnson, King Clyde B. Yabut, Michael MacCoss, Trisha N. Davis, Eric Muller, Andrej Sali, David A. Agard
bioRxiv 311027; doi: https://doi.org/10.1101/311027

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