The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome

Rohrwild, Markus; Pfeifer, Günter; Santarius, Ute; Müller, Shirley A.; Huang, Hsin‐Chun; Engel, Andréas; Baumeister, Wolfgang; Goldberg, Alfred L.

doi:10.1038/nsb0297-133

Cited by 182 publications

(138 citation statements)

References 44 publications

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“…Instead, HslU could shuttle between states of high and low affinity for HslV and deliver substrates in the process. In contrast, the EM images (13), interpreted in the light of the crystal structures of both components, indicate binding of HslU to HslV with the I domains distal to HslV (this will be referred to as the EM mode of docking).…”

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confidence: 99%

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Mutational studies on HslU and its docking mode with HslV

Song

Hartmann

Ramachandran

et al. 2000

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

133

124

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HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition͞translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the ''sensor arginine'' (R393) and the ''arginine finger'' (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

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“…Additionally, and most importantly, different docking modes are observed in the crystal structure and in electron microscopy (EM) images (10,13). In the crystal structure, HslU docks to HslV with the I domains pointing toward the HslV particle (this will be referred to as the x-ray mode of docking) (10).…”

mentioning

confidence: 99%

Mutational studies on HslU and its docking mode with HslV

Song

Hartmann

Ramachandran

et al. 2000

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

133

124

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“…Monomers include one (ClpX, ClpY) or two (ClpA, ClpB) highly conserved nucleotide binding domains (17), referred to as AAA domains (7,18). Crystal structures (19)(20)(21) and oligomeric models (22,23) based on electron microscopy images (24,25) reveal the presence of a central channel with a diameter of approximately 9-15 Å at the narrowest point. The channel is occupied by diaphragm-forming loops (one per AAA domain), which contain a conserved motif consisting of an aromatic-hydrophobic dipeptide flanked by glycine amino acids.…”

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confidence: 99%

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Kravats

Jayasinghe²,

Stan³

2011

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

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Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarsegrained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.AAA+ protease | molecular machine | protein translocation I ntracellular regulatory mechanisms for selective destruction of proteins and disassembly of protein aggregates are critical for the maintenance of vital cellular functions (1). Clp macromolecular machines, found in all domains of life from prokaryotes to multicellular eukaryotes (2), perform such protein quality control using powerful ATPase components (3-6) that effect protein unfolding and translocation through narrow pores. Clp (caseinolytic protease) ATPases, are members of the AAA+ (ATPases associated with diverse cellular activities) superfamily of proteins (7, 8) responsible for a variety of functions including intracellular transport, DNA replication and repair, and transcription regulation (9-11). The best understood Clp ATPases are ClpA; ClpX and ClpY (HslU) (12), which enable protein degradation by associating with peptidase subunits ClpP and ClpQ (HslV); and ClpB, which promotes disaggregation (13-15).Functional forms of Clp ATPases are homohexameric assemblies that form in the presence of ATP (16). Monomers include one (ClpX, ClpY) or two (ClpA, ClpB) highly conserved nucleotide binding domains (17), referred to as AAA domains (7,18). Crystal structures (19-21) and oligomeric models (22, 23) based on electron microscopy images (24, 25) reveal the presence of a central channel with a diameter of approximately 9-15 Å at the narrowest point. The channel is occupied by diaphragm-forming loops (one per AAA domain), which contain a conserved motif consisting of an aromatic-hydrophobic dipeptide flanked by glycine amino acids. These loops are implicated in the ti...

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“…However, the orientation of the HslU hexamers with respect to the HslV double-hexamer rings is opposite in the two models. The most compact organization, with the so-called intermediate domains (or I-domains) pointing outwards, as described by Sousa et al [7 •• ], is favored by electron microscopy studies [8] and small-angle scattering data. Sousa et al [7 •• ] suggest that the I-domains serve to bind substrates and lead them into the interior cavity of the complex, where the active sites are located (Figure 1).…”

Section: Self-compartmentalizing Proteasesmentioning

confidence: 99%

Novel proteases: common themes and surprising features

Vandeputte-Rutten

2002

Current Opinion in Structural Biology

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The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome

Cited by 182 publications

References 44 publications

Mutational studies on HslU and its docking mode with HslV

Mutational studies on HslU and its docking mode with HslV

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Novel proteases: common themes and surprising features

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