Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Rui Huang; Zev A Ripstein; Rafal Augustyniak; Michal Lazniewski; Krzysztof Ginalski; Lewis E Kay; John L Rubinstein

doi:10.1073/pnas.1603980113

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):E4190-9. doi: 10.1073/pnas.1603980113. Epub 2016 Jul 11.

Authors

Rui Huang¹, Zev A Ripstein², Rafal Augustyniak¹, Michal Lazniewski³, Krzysztof Ginalski⁴, Lewis E Kay⁵, John L Rubinstein⁶

Affiliations

¹ Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8;
² Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4;
³ Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland; Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland;
⁴ Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland;
⁵ Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca.
⁶ Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7 kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca.

Abstract

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.

Keywords: VAT; cryo-EM; methyl-TROSY NMR; proteasome; protein dynamics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Archaeal Proteins / chemistry*
Archaeal Proteins / genetics
Cryoelectron Microscopy
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Thermoplasma / enzymology
Thermoplasma / genetics
Valosin Containing Protein / chemistry*
Valosin Containing Protein / genetics

Substances

Archaeal Proteins
Valosin Containing Protein
vat protein, Thermoplasma acidophilum