MassIVE MSV000085155

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

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Description

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) six-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 mass spectrometry study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the wild-type protein. We utilized ADP and its analogues (TNP-ADP and mant-ADP), and a non-hydrolyzable ATP analogue (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 non-specific nucleotide binding. This approach led to the unambiguous finding that a wild-type PAN hexamer carried, from expression host six tightly bound ADP molecules that could be exchanged for ADP and ATP analogues. While the Walker A mutant did not bind ADP analogues, 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. [doi:10.25345/C5R10P] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: proteasome, AAA+ ATPase, nucleotide binding, stoichiometry, native mass spectrometry, cooperativity

Contact

Principal Investigators:
(in alphabetical order) 		H. Ewa Witkowska, UCSF, United States
Submitting User: halinaewa

Publications

Yadong Yu, Haichuan Liu, Zanlin Yu, H. Ewa Witkowska, Yifan Cheng.
Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry.
mcp.RA120.002067; https://doi.org/10.1074/mcp.RA120.002067.

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