# Mapping Hsp104 interactions using cross‐linking mass spectrometry

## TL;DR

This paper uses cross-linking mass spectrometry to study how ATP affects the structure of Hsp104 and its interactions with proteins.

## Contribution

A new analytical pipeline for XL-MS is developed to distinguish intra- and inter-subunit contacts in Hsp104.

## Key findings

- Cross-links reveal conformational changes in ATP-hydrolysis competent Hsp104.
- Contacts between Hsp104 and PCSK9 suggest interactions during translocation.
- XL-MS experiments help interpret molecular machine conformation and protein binding.

## Abstract

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.

This study examines how cross‐linking mass spectrometry can be utilized to analyze ATP‐induced conformational changes in Hsp104 and its interactions with substrates. We developed an analytical pipeline to distinguish between intra‐ and inter‐subunit contacts within the hexameric homo‐oligomer and discovered contacts between Hsp104 and a selected protein, PCSK9. These experiments enhance our understanding of how Hsp104 undergoes conformational changes.