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

Elnatan, Daniel; Betegon, Miguel; Liu, Yanxin; Ramelot, Theresa; Kennedy, Michael A.; Agard, David A.

doi:10.7554/elife.25235

Cited by 74 publications

(118 citation statements)

References 66 publications

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“…The unitary Hill coefficient for both ATP and magnesium likely reflects the obligatory sequential (noncooperative) nature of ATP hydrolysis previously observed (20). Identifying where calcium binds would greatly facilitate understanding how calcium imparts an apparent cooperativity in TRAP1 ATPase activity, which has never been observed in other Hsp90 homologs.…”

Section: Resultssupporting

confidence: 54%

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Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

Elnatan

Agard

2018

Journal of Biological Chemistry

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The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor–associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients.

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Section: Resultssupporting

confidence: 54%

“…4) (20). As the primary client-binding site maps to the region of maximal asymmetry, this suggests a strong coupling between the asymmetry flip and client remodeling.…”

Section: Introductionmentioning

confidence: 99%

Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

Elnatan

Agard

2018

Journal of Biological Chemistry

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“…ER and mitochondrial equivalents of these cochaperones are not present, but both GRP94 and Trap1 contain a significant pre-N domain. It is possible that, in the pared-down chaperoning world of the organellar compartments, the complexity of cochaperone regulation is replaced by intrinsic regulation via unique chaperone extensions, as in GRP94, or other structural adaptations such as the asymmetry found in Trap1 (Elnatan et al, 2017; Lavery et al, 2014). …”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Analysis of GRP94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site

et al. 2017

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Summary Hsp90 chaperones undergo ATP-driven conformational changes during the maturation of client proteins, populating a “closed” state upon ATP binding in which the N-terminal domains of the homodimer form a second inter-protomer dimer interface. A structure of GRP94, the endoplasmic reticulum hsp90, in a closed conformation has not been described, and the determinants that regulate closure are not well understood. Here, we determined the 2.6 Å structure of AMPPNP-bound GRP94 in the closed dimer conformation. The structure includes the pre-N domain, a region preceding the N-terminal domain that is highly conserved in GRP94, but not in other hsp90s. We show that the GRP94 pre-N domain is essential for client maturation, and identify the pre-N domain as an important regulator of ATPase rates and dimer closure. The structure also reveals a GRP94:polypeptide interaction that partially mimics a client-bound state. The results provide structural insight into the ATP-dependent client maturation process of GRP94.

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“…The constructs for human TRAP1 (hsTRAP1) fused either SpyTag or Spy Catchter were the same as previously used 24 . Proteins were expressed in E. coli BL21-star (DE3) cells.…”

Section: Protein Purificationmentioning

confidence: 99%

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

Wang

Liu

et al. 2019

Preprint

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Despite their great potential to facilitate rapid preparation of quite impure samples, affinity gridshave not yet been widely employed in single particle cryo-EM. Here, we chemically functionalize graphene oxide coated grids and use a highly specific covalent affinity tag system. Importantly, our polyethylene glycol spacer keeps particles away from the air-water interface and graphene oxide surface, protecting them from denaturation or aggregation and permits high-resolution reconstructions of small particles. MainOwing to a series of technological breakthroughs, cryo-electron microscopy (cryo-EM) is fast becoming the method of choice for determining protein structures at a near-atomic or atomic resolution 1, 2 . By contrast with the rapidity of data collection and processing, sample preparation remains slow and, in many cases, has become rate-limiting. Cryo-EM specimens are typically prepared by depositing purified proteins onto cryo-EM grids, which are usually a metal grid covered with continuous perforated carbon or gold films.

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

Cited by 74 publications

References 66 publications

Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

Structural and Functional Analysis of GRP94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

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