Elnatan, D. | bioRxiv Science

bioRxiv2018

Modulation of mitochondrial Hsp90 (TRAP1) ATPase activity by calcium and magnesium

The Hsp90 protein family are ATP-dependent molecular chaperones that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotic cells have organelle-specific Hsp90 paralogs that are adapted to each unique sub-cellular environment. The mitochondrial Hsp90, TRAP1, supports the folding and activity of electron transport components and is increasingly being appreciated as a critical player in mitochondrial signaling. It is well known that calcium plays an important regulatory role in mitochondria and can even accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we find that calcium can replace the requirement for magnesium to support TRAP1 ATPase activity. Using anomalous x-ray diffraction, we reveal a novel calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP -phosphate and completely distinct from the magnesium site adjacent to the {beta} and {gamma}-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, is non-cooperative, whereas calcium binding results in cooperative ATP hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggest a mechanism for the cooperative behavior. Owing to the cooperativity, at high ATP concentrations, ATPase activity is higher with calcium, whereas the converse is true at low ATP concentrations. Integrating these observations, we propose a model where the divalent cations choice can control switching between non-cooperative and cooperative TRAP1 ATPase mechanisms in response ATP concentrations. This may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle.

biochemistry↗

bioRxiv2017

Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate \"client\" proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly-strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling.

molecular biology↗