Mapping Hsp104 structure and substrate interactions using crosslinking mass spectrometry

Molecular machines from the AAA+ family play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in conformation that underlie their specialized functions. Furthermore, complexes between these machines and substrates begin to explain their mechanism of activity. Here we explore how crosslinking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP and how substrates are associated. We applied a panel of crosslinking reagents to produce high-resolution crosslinking maps 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 crosslinks that break the asymmetry that are only present when ATP is bound and are absent in an ATP-binding deficient mutant. Finally, we identify contacts between Hsp104 and a model substrate to identify contacts on the central channel of Hsp104 across the length of the substrate indicating that we have trapped interactions consistent with translocation of the substrate. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to substrates even in the context of homo-oligomeric assemblies.