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Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry

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AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

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Highlights

  • All six binding sites in PANWT are occupied by ADP- or ATP-type nucleotides.

  • PANKA Walker A mutant substoichiometrically binds ATP- but not ADP-type nucleotides.

  • PAN hexamer dissociation of the solution origin characteristics was observed in MS.

  • We posit that the PAN hexamer dissociation proceeds within the ESI droplets.

proteasome
AAA+ ATPase
nucleotide binding
stoichiometry
native mass spectrometry
cooperativity
mass spectrometry
Archaebacteria*
electron microscopy
macromolecular complex analysis
non-covalent interaction MS*
protea-some

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This article contains supplemental data

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.