Mapping Hsp104 interactions using cross‐linking mass spectrometry

Westphal, Kinga; Michalska, Karolina; Joachimiak, Andrzej; Joachimiak, Lukasz A.

doi:10.1002/2211-5463.70007

Mapping Hsp104 interactions using cross‐linking mass spectrometry

Journal Article · Mon Feb 24 00:00:00 EST 2025 · FEBS Open Bio

DOI:https://doi.org/10.1002/2211-5463.70007· OSTI ID:2573052

^[1]; Michalska, Karolina ^[2]; ^[2]; ^[3]

Univ. of Texas Southwestern Medical Center, Dallas, TX (United States); Warsaw Univ. of Technology (Poland)
Univ. of Chicago, IL (United States); Argonne National Laboratory (ANL), Argonne, IL (United States)
Univ. of Texas Southwestern Medical Center, Dallas, TX (United States); Argonne National Laboratory (ANL), Argonne, IL (United States)

Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross-linking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross-linking reagents to produce cross-linking maps of Hsp104 and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify cross-links that break the asymmetry that is present in Hsp104 in an ATP-hydrolysis competent conformation but is absent in an ATP-hydrolysis-defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo-oligomeric assemblies enabling coupling state-of-the-art modeling approaches with XL-MS.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2573052

Journal Information:: FEBS Open Bio, Journal Name: FEBS Open Bio Journal Issue: 6 Vol. 15; ISSN 2211-5463

Publisher:: Federation of European Biochemical SocietiesCopyright Statement

Country of Publication:: United States

Language:: English

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References (57)

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