Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions

Duran, Elizabeth C.; Weaver, Clarissa L.; Lucius, Aaron L.

doi:10.3389/fmolb.2017.00054

Cited by 28 publications

(20 citation statements)

References 128 publications

(229 reference statements)

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“…The D1 and D2 rings of the ClpB and Hsp104 protein-remodeling machines share strong sequence and structural homology with those of ClpA ( 8 , 22 , 47 ). However, based on the findings in this study, coordination of ATPase modules within and between the D1 and D2 rings appears to be different in ClpA ( 18 , 48 – 51 ).…”

Section: Discussionmentioning

confidence: 99%

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

Zuromski

Sauer

Baker

2020

Proc. Natl. Acad. Sci. U.S.A.

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ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.

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Section: Discussionmentioning

confidence: 99%

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

Zuromski

Sauer

Baker

2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Hsp104 | HX MS | HDX-MS | AAA+ | molecular machine T he homohexameric Hsp104 disaggregase is a highly studied member of the AAA+ superfamily of molecular machines, which perform myriad biological functions (1)(2)(3)(4)(5). Hsp104 transduces the favorable energy of ATP binding and hydrolysis through local and global structure changes (6,7) to drive the forceful energy-requiring disaggregation of substrate proteins by drawing them into and sometimes through its narrow axial pore (8)(9)(10)(11).…”

mentioning

confidence: 99%

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Lin

Mayne

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

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“…In the tomato microspores, ten of the 35 HS-up-regulated SCPs are in the heat acclimation process. These tomato proteins include Hsp20, (Solyc07g045610, Solyc06g076540, Solyc06g076520, Solyc09g059210, [24,25] and the heat-inducible ClpB chaperone class [26,27]. These HSPs serve as chaperones for protein folding and refolding and thus have a critical role in maintaining proteome homeostasis throughout microspore and pollen development [13].…”

Section: Discussionmentioning

confidence: 99%

Identification of Heat-Induced Proteomes in Tomato Microspores Using LCM- Proteomics Analysis

H¹,

Zhu²,

Rangu³

et al. 2018

SCB

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Pollen development is highly susceptible to heat stress (HS) and the production of inviable pollen causes reduction in seed-and fruit-set in plants. This study was carried out to identify HS-induced pollen proteins and the associated biological processes in tomato (Solanum lycopersicum). Tomato 'Micro-Tom' plants were incubated under 32°C//22°C (day/night, 12/12 h) for two weeks for heat treatment, and the non-treated control plants were incubated for the same time period at 25°C /22°C. Flower buds of 5 mm in length were confirmed to contain the heat sensitive uninucleate microspores. Pollen cells were harvested using laser capture microdissection (LCM) and protein was extracted using a one-step method under high pressure and vacuum. Approximately 60,000 LCMharvested microspore cells yielded about 18-20 μg proteins. The tandem mass tags (TMT) proteomics analysis identified a total of 6018 proteins, 4784 proteins were quantified, 37 proteins were identified as HS up-regulated significantly changed proteins (SCPs), and 83 proteins as HS down (dn)-regulated SCPs. Further analysis using the plant MetGenMap system showed that the HS up-regulated SCPs were enriched in the heat acclimation, pollen wall formation, protein folding/refolding gene ontology (GO) biological processes, and the HS dn-regulated SCPs were placed in the carbohydrate catabolism and de-novo protein biosynthesis GO terms. Biological processes such as mitosis, resistance to oxidative stresses, and carbohydrate and lipid metabolic processes contain both the HS up-, and dn-regulated SCPs. These results indicate that the LCM-TMT proteomics workflow is highly efficient in the identification of HS-induced pollen proteomes. These HS induced SCPs will be used for exploring heat tolerance of tomato pollens. The proteomics data are available via ProteomeXchange with identifier PXD010218.

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Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions

Cited by 28 publications

References 128 publications

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Identification of Heat-Induced Proteomes in Tomato Microspores Using LCM- Proteomics Analysis

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