scholarly journals Structure of the Cdc48 segregase in the act of unfolding an authentic substrate

Science ◽  
2019 ◽  
Vol 365 (6452) ◽  
pp. 502-505 ◽  
Author(s):  
Ian Cooney ◽  
Han Han ◽  
Michael G. Stewart ◽  
Richard H. Carson ◽  
Daniel T. Hansen ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Substrate Binding ◽  
Adaptor Protein ◽  
Biological Pathways ◽  
Protein Substrates ◽  
Adenosine Triphosphatases ◽  
Binding Residues ◽  
Aaa Atpases ◽  
Helical Configuration ◽  
Resolution Structure

The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom–resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases.

scholarly journals Oligomerization of a molecular chaperone modulates its activity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Tomohide Saio ◽  
Soichiro Kawagoe ◽  
Koichiro Ishimori ◽  
Charalampos G Kalodimos
Keyword(s):  
Binding Sites ◽  
Substrate Binding ◽  
Structural Data ◽  
Trigger Factor ◽  
Unfolded Proteins ◽  
Protein Substrates ◽  
Native Proteins ◽  
Dimeric Interface ◽  
Substrate Binding Sites ◽  
Resolution Structure

Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell.

scholarly journals Proteasomes: unfoldase-assisted protein degradation machines

2019 ◽  
Vol 401 (1) ◽  
pp. 183-199 ◽  
Author(s):  
Parijat Majumder ◽  
Wolfgang Baumeister
Keyword(s):  
Stress Response ◽  
Molecular Machines ◽  
Molecular Architecture ◽  
Core Particle ◽  
Protein Substrates ◽  
Macromolecular Assemblies ◽  
Current State ◽  
Intracellular Proteins ◽  
Domains Of Life ◽  
Aaa Atpases

Abstract Proteasomes are the principal molecular machines for the regulated degradation of intracellular proteins. These self-compartmentalized macromolecular assemblies selectively degrade misfolded, mistranslated, damaged or otherwise unwanted proteins, and play a pivotal role in the maintenance of cellular proteostasis, in stress response, and numerous other processes of vital importance. Whereas the molecular architecture of the proteasome core particle (CP) is universally conserved, the unfoldase modules vary in overall structure, subunit complexity, and regulatory principles. Proteasomal unfoldases are AAA+ ATPases (ATPases associated with a variety of cellular activities) that unfold protein substrates, and translocate them into the CP for degradation. In this review, we summarize the current state of knowledge about proteasome – unfoldase systems in bacteria, archaea, and eukaryotes, the three domains of life.

scholarly journals Structure and mechanism of the ESCRT pathway AAA+ ATPase Vps4

2019 ◽  
Vol 47 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Han Han ◽  
Christopher P. Hill
Keyword(s):  
Cell Biology ◽  
Cellular Membrane ◽  
Structural Features ◽  
Transport Pathways ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Protein Substrates ◽  
Membrane Fission ◽  
Aaa Atpases ◽  
Equivalent Mechanism

Abstract The progression of ESCRT (Endosomal Sorting Complexes Required for Transport) pathways, which mediate numerous cellular membrane fission events, is driven by the enzyme Vps4. Understanding of Vps4 mechanism is, therefore, of fundamental importance in its own right and, moreover, it is highly relevant to the understanding of many related AAA+ ATPases that function in multiple facets of cell biology. Vps4 unfolds its ESCRT-III protein substrates by translocating them through its central hexameric pore, thereby driving membrane fission and recycling of ESCRT-III subunits. This mini-review focuses on recent advances in Vps4 structure and mechanism, including ideas about how Vps4 translocates and unfolds ESCRT-III subunits. Related AAA+ ATPases that share structural features with Vps4 and likely utilize an equivalent mechanism are also discussed.

scholarly journals Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Valentina Peleh ◽  
Emmanuelle Cordat ◽  
Johannes M Herrmann
Keyword(s):  
Disulfide Bonds ◽  
Protein Import ◽  
Protein Translocation ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Intermembrane Space ◽  
Cysteine Motif ◽  
Mitochondrial Intermembrane Space ◽  
Necessary And Sufficient ◽  
Yeast Mutants

Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism.

scholarly journals The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation

1999 ◽  
Vol 145 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Alessio Merlin ◽  
Wolfgang Voos ◽  
Ammy C. Maarse ◽  
Michiel Meijer ◽  
Nikolaus Pfanner ◽  
...  
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
J Proteins

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Δ18 in addition to the endogenous wild-type Tim44. Tim44Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

scholarly journals Small Heat Shock Protein IbpB Acts as a Robust Chaperone in Living Cells by Hierarchically Activating Its Multi-type Substrate-binding Residues

2013 ◽  
Vol 288 (17) ◽  
pp. 11897-11906 ◽  
Author(s):  
Xinmiao Fu ◽  
Xiaodong Shi ◽  
Linxiang Yin ◽  
Jiafeng Liu ◽  
Keehyoung Joo ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Substrate Binding ◽  
Small Heat Shock Protein ◽  
Living Cells ◽  
Binding Residues

Catalytic Roles of Substrate-Binding Residues in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase

Biochemistry ◽  
2014 ◽  
Vol 53 (16) ◽  
pp. 2661-2671 ◽  
Author(s):  
Koichi Mori ◽  
Toshihiro Oiwa ◽  
Satoshi Kawaguchi ◽  
Kyosuke Kondo ◽  
Yusuke Takahashi ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Coenzyme B12 ◽  
Binding Residues

scholarly journals Structures and operating principles of the replisome

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. eaav7003 ◽  
Author(s):  
Yang Gao ◽  
Yanxiang Cui ◽  
Tara Fox ◽  
Shiqiang Lin ◽  
Huaibin Wang ◽  
...  
Keyword(s):  
Replication Fork ◽  
Model System ◽  
Molecular Organization ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Dna Strands ◽  
Leading Strand ◽  
Dna Strand ◽  
Aaa Atpases ◽  
Lagging Strand

Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

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