Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Hsp104 is a AAA+ molecular machine crucial for disaggregating proteins trapped in amorphous aggregates and amyloids.
• High-resolution cryo-electron microscopy (cryo-EM) has provided structural insights into Hsp104.
• Understanding the dynamic conformational changes and nucleotide-dependent mechanisms of Hsp104 is essential for its function.

Purpose of the Study:

• To characterize the functionally active and inactive elements of Hsp104 using hydrogen exchange mass spectrometry (HX MS).
• To elucidate the dynamic conformational cycling of Hsp104 during ATP hydrolysis.
• To determine the response of Hsp104 structural elements to different bound adenosine nucleotides.

Main Methods:

• Hydrogen exchange mass spectrometry (HX MS) was employed to analyze Hsp104 structure and dynamics.
• HX MS data were correlated with existing cryo-EM structural data.
• Functional states were probed using various adenosine nucleotide analogs (ADP, ATP, AMPPNP, ATPγS).

Main Results:

• HX MS confirmed a noncanonical interprotomer interface as a marker for the Hsp104 hexamer's spiraled conformation and measured its conformational cycling.
• The study revealed the distinct responses of Hsp104 structural elements to different bound nucleotides, particularly the Walker A segments.
• Binding of ATPγS induced a global open-to-closed/extended transition, leading to pore constriction and increased structural integrity of pore-related loops, indicative of substrate translocation energy transfer.

Conclusions:

• Hsp104 undergoes rapid, nucleotide-dependent conformational cycling essential for its disaggregase activity.
• The structural changes, particularly pore constriction upon ATP analog binding, are directly linked to the energy-requiring process of substrate protein translocation.
• ATP hydrolysis to ADP facilitates Hsp104 relaxation to an open state, preparing it for the next cycle of protein rescue.