The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease

Ishikawa, Takashi; Maurizi, Michael R.; Steven, Alasdair C.

doi:10.1016/j.jsb.2003.11.018

Cited by 48 publications

(52 citation statements)

References 51 publications

(65 reference statements)

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“…This might contribute to a relative lengthening of the AMIB tail. There is a precedent for the grafting of additional domains onto different sites on a conserved framework in the Clp ATPases (36). The high sequence similarity of the GPA2 regions of AMIB and AMIC (70% identity) supports this notion, as does our observation that AMIC GPA1 and GPA2 are in close proximity.…”

Section: Discussionsupporting

confidence: 72%

Subdomain organization of theAcanthamoebamyosin IC tail from cryo-electron microscopy

Ishikawa¹,

Cheng²,

Liu³

et al. 2004

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

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Acanthamoeba myosin IC (AMIC) is a single-headed myosin comprised of one heavy chain (129 kDa) and one light chain (17 kDa). The heavy chain has head, neck (light chain-binding), and tail domains. The tail consists of four subdomains: a basic region (BR) (23 kDa) and two Gly͞Pro͞Ala-rich (GPA) regions, GPA1 (6 kDa) and GPA2 (15 kDa), flanking an Src homology 3 region (6 kDa). Although the AMIC head is similar in sequence, structure, and function (ATPase motor) to other myosin heads, the organization of the tail has been less clear as has its function beyond an assumed role in binding interaction partners, e.g., the BR has a membrane affinity and the GPA components bind F-actin in an ATP-independent manner. To investigate the spatial arrangement of subdomains in the tail, we have used cryo-electron microscopy and image reconstruction to compare actin filaments decorated with WT AMIC and tail-truncated mutants of various lengths. The BR forms an oval-shaped feature, Ϸ40 Å long, that diverges obliquely from the head, extending azimuthally around the actin filament and toward its barbed end. GPA2 and GPA1 are located together on the inner (actin-proximal) side of the tail, close enough to act in concert in binding the same or another actin filament. The outer face of the BR is strategically exposed for membrane or vesicle binding.

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Section: Discussionsupporting

confidence: 72%

Subdomain organization of theAcanthamoebamyosin IC tail from cryo-electron microscopy

Ishikawa¹,

Cheng²,

Liu³

et al. 2004

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

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“…A recent cryo-electron microscopy study comparing intact ClpA with an N-domain-deleted version suggests that the N-domains rise in the axial direction out of the body of the cylinder, emerging from the D1 AAAϩ domain (34). Simulations of this electron microscopy data suggest that the N-domains are very mobile in their position atop the cylinder, consistent with what appears from structural studies to be attachment to the D1 domain via a flexible linker segment (amino acids 143-167).…”

mentioning

confidence: 99%

Roles of the N-domains of the ClpA Unfoldase in Binding Substrate Proteins and in Stable Complex Formation with the ClpP Protease

Hinnerwisch

Reid

Fenton

et al. 2005

Journal of Biological Chemistry

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The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of ϳ150 amino acids at the top of the cylinder followed by two AAA؉ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins ( Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.

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“…Such methods include cryo EM and SAXS. Cryo EM has revealed flexibility in the N-domains of E. coli AAA + protein ClpA (Ishikawa et al, 2004) and SAXS experiments have shown large conformational changes in NtrC1 (Chen et al, 2010). Although in some cases conformational changes observed with different methods do not completely agree, it is widely accepted that AAA + proteins undergo dynamic movements during their catalytic cycle.…”

Section: Conformational Changes In Aaa + Proteinsmentioning

confidence: 99%

IBMPFD and p97, the Structural and Molecular Basis for Functional Disruption

Tang¹,

Xia²

2012

Neuromuscular Disorders

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The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease

Cited by 48 publications

References 51 publications

Subdomain organization of theAcanthamoebamyosin IC tail from cryo-electron microscopy

Subdomain organization of theAcanthamoebamyosin IC tail from cryo-electron microscopy

Roles of the N-domains of the ClpA Unfoldase in Binding Substrate Proteins and in Stable Complex Formation with the ClpP Protease

IBMPFD and p97, the Structural and Molecular Basis for Functional Disruption

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