Activating Compound | Comment | Organism | Structure |
---|---|---|---|
HSP70 | - |
Escherichia coli | |
additional information | mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. ClpB activation requires two signals: (1) interaction with the Hsp70 partner chaperone and (2) binding to substrate protein | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
E279A/K476C/E678A | site-directed mutagenesis, the mutant shows a reduced number of subunits | Escherichia coli |
K476C | site-directed mutagenesis, the mutation weakens the dynamic interaction between M-domain and AAA1 ring resulting in M-domain dissociation and persistent, Hsp70-independent derepression of ClpB ATPase activity. Consequently, the ATPase activation by substrate is much stronger than in wild-type ClpB, and mutant ClpB-K476C has increased protein disaggregation activity, linked to its ability to unfold stable domains, an activity not observed for wild-type ClpB. ATP concentrations at half-maximal ATP hydrolysis rates of ClpB-K476C drop to 1.4 mM in the presence of substrate, compared with 4.3 mM for wild-type ClpB in the presence of substrate indicating that only the fully two-step activated state of ClpB reaches high ATPase activity at physiological ATP concentrations, which coincides with decreased cooperativity. Substrate-bound ClpB-K476C structures reveal large displacements of AAA2 pore loops | Escherichia coli |
Y251A/K476C | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to wild-type enzyme | Escherichia coli |
Y251A/K476C/Y653A | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to wild-type enzyme | Escherichia coli |
Y653A/K476C | site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to wild-type enzyme | Escherichia coli |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
additional information | coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | cooperative mode of ATP hydrolysis | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P63284 | - |
- |
Subunits | Comment | Organism |
---|---|---|
hexamer | - |
Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
ATPase | - |
Escherichia coli |
ClpB | - |
Escherichia coli |
ClpB disaggregase | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
malfunction | substrate-triggered decrease in cooperativity of mutant ClpB-K476C ATPase activity. The K476C mutation weakens the dynamic interaction between M-domain and AAA1 ring resulting in M-domain dissociation and persistent, Hsp70-independent derepression of ClpB ATPase activity. Consequently, the ATPase activation by substrate is much stronger than in wild-type ClpB, and mutant ClpB-K476C has increased protein disaggregation activity, linked to its ability to unfold stable domains, an activity not observed for wild-type ClpB. ATP concentrations at half-maximal ATP hydrolysis rates of ClpB-K476C drop to 1.4 mM in the presence of substrate, compared with 4.3 mM for wild-type ClpB in the presence of substrate indicating that only the fully two-step activated state of ClpB reaches high ATPase activity at physiological ATP concentrations, which coincides with decreased cooperativity. Substrate-bound ClpB-K476C structures reveal large displacements of AAA2 pore loops. Comparisons of substrate binding structures of wild-type and mutant enzymes, overview | Escherichia coli |
additional information | disaggregase ClpB contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism. Residue Lys476 is part of a conserved salt bridge network that regulates the dynamic interaction between M-domain and AAA1 ring. Comparisons of substrate binding structures of wild-type and mutant enzymes, overview | Escherichia coli |