Molecular & Cellular Proteomics
ResearchStoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry
Graphical Abstract
Highlights
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All six binding sites in PANWT are occupied by ADP- or ATP-type nucleotides.
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PANKA Walker A mutant substoichiometrically binds ATP- but not ADP-type nucleotides.
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PAN hexamer dissociation of the solution origin characteristics was observed in MS.
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We posit that the PAN hexamer dissociation proceeds within the ESI droplets.
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Funding and additional information—This study has been supported in part by a grant from National Institutes of Health (R01GM082893) and a grant from UCSF Program for Breakthrough Biomedical Research (Opportunity Award in Basic Science) to YC and by grants from NIGMS (R01GM51923-13). YC is an Investigator of Howard Hughes Medical institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
Abbreviations—The abbreviations used are:
- PAN
proteasome activating nucleotidase
- MS
mass spectrometry
- ESI
electrospray ionization
- TNP-ADP
2'-(or-3')-O-(trinitrophenyl)adenosine 5'-diphosphate
- mant-ADP
2'-(or-3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate
- AMP-PNP
adenosine 5'-(β,γ-imido)triphosphate
- ATPγS
adenosine 5'-O-(3-thio)triphosphate
- Rpt
regulatory particle of triple-ATPase.
Present address for Yadong Yu: TrueBinding Inc. 1140A O'Brien Dr., Menlo Park, CA 94205, USA.
Present address for Haichuan Liu: Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA.
Author contributions—Y.Y. conceived the project, designed, and performed experiments, and analyzed data. Z.Y. performed some biochemical experiments. H.L. performed all MS experiments and processed the spectra. H.E.W. analyzed the mass spectra. Y.C. supervised the research. All authors contributed to manuscript writing.