Information on EC 3.6.3.51 - mitochondrial protein-transporting ATPase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.6.3.51
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RECOMMENDED NAME
GeneOntology No.
mitochondrial protein-transporting ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
transmembrane transport
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (mitochondrial protein-importing)
A non-phosphorylated, non-ABC (ATP-binding cassette) ATPase involved in the transport of proteins or preproteins into mitochondria using the TIM protein complex. (TIM is the protein transport machinery of the inner mitochondrial membrane that contains three essential Tim proteins: Tim17 and Tim23 are thought to build a preprotein translocation channel while Tim44 interacts transiently with the matrix heat-shock protein Hsp70 to form an ATP-driven import motor.)
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
African green monkey
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Manually annotated by BRENDA team
strain WB
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Manually annotated by BRENDA team
strain WB
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain BY4743
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Manually annotated by BRENDA team
strain PK81, PK82 and PK83
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Manually annotated by BRENDA team
strain YPH499
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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temperature-sensitive mutants of mitochondrial Hsp70, affected in either ATP-binding and hydrolysis or Tim44 interaction, display defects in matrix protein import and concomitant TOM/TIM23-supercomplex formation when import reactions were performed under non-permissive conditions
metabolism
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for the ATP-dependent translocation of preproteins into the aqueous matrix, TIM23CORE cooperates with the presequence translocase-associated import motor (PAM) that is composed of mitochondrial heat shock protein 70 (mtHsp70) and five co-chaperones that differentially regulate the import-driving ATPase activity of mtHsp70 and the assembly status of PAM: Tim44, Pam16, Pam17, Pam18, and Mge1. The biogenesis of presequence-carrying polytopic inner membrane proteins requires a close cooperation of the TIM23/PAM machinery with the export translocase Oxa1. Therefore, the TIM23 complex acts as an intricate multipurpose molecular machine in which the coupling of distinct partner proteins to TIM23CORE generates functional specificity
physiological function
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in mitochondria, matrix translocation of polypeptides depends on the ATP-powered mitochondrial Hsp70 (mtHsp70), the catalytic constituent of the presequence translocase-associated motor (PAM), at the trans side of the protein-conducting channel. mtHsp70 exists in a soluble pool mediating matrix protein folding and a TIM23-associated pool generating precursor velocity over the inner membrane. During precursor transport, a translocation intermediate is established in which the motor-associated, unfolded precursor simultaneously spans the TOM and TIM23 complexes thereby generating a mitochondrial contact site. For both activities, the soluble co-chaperone Mge1 stimulates ADP/ATP exchange. To drive precursor translocation, the Hsp70-import motor associates with the protein-conducting channel of the TIM23 complex, the ATPase cycle of Hsp70 is regulated in the context of a translocating polypeptide chain. The presence of all Hsp70 co-chaperones at the import channel is not sufficient to promote matrix protein import, instead a recharging of the active translocase with Pam18 is required for motor activity. Thus, a replenishment cycle of co-chaperones at the TIM23 complex is an integral part of Hsp70’s ATPase cycle at the channel exit site and essential to maintain motor-driven mitochondrial protein import. The association of the membrane-bound co-chaperones with the Tim23 channel unit is a prerequisite for the spatially controlled mtHsp70 regulation in mitochondria. Pam18 needs to be recharged at the translocase in order to maintain progressive mtHsp70 activity, such a replenishment cycle of the translocase’s co-chaperone Pam18 drives the ATP hydrolysis of the import motor in the precursor-occupied translocase and thus precursor transport along the presequence pathway, overview
additional information
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quantitative analysis of mitochondrial protein complexes, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + protein/out
ADP + phosphate + protein/in
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + protein/out
ADP + phosphate + protein/in
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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the presequence-cleaved mature forms of the recombinant fusion proteins with a 50 or 60 residue spacer sequence are proteinase K-resistant, whereas those with a 70 or 80 residue spacer sequence are proteinase K-sensitive
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DnaJ
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bacterial co-chaperone stimulates ATPase activity of mtHsp70 5fold, that of DnaK 20fold
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fluorescein-CALLQSRLLLSAPRRAAATARY
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similar affininty of mtHsp70 and Dnak at 25°C
GrpE
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bacterial co-chaperone stimulates ATPase activity of mtHsp70 5fold, that of DnaK 20fold
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GrpEL1
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the proposed nucleotide exchange factor of the human mtHsp70 machine. To access the nucleotide relase of human GrpEL1, a single turnover ATPase assay is performed to monitor the hydrolysis of the prebound mtHsp70-ATP complex of the wild-type protein in the presence of GrpEL1. Human GrpEL1 shows robust nucleotide exchange activity in the presence of excess unlabeled ATP, thus inhibiting the maximum ATP hydrolysis of wild-type mtHsp70
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H2O2
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stimulates association of mtHsp70 with wild-type DJ-1
Hsp40-related J domain protein DJA1
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Hsp40-related J domain protein DJA2
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Hsp40-related J domain protein DJA4
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human escort protein
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Hep, stimulates the activity of mtHsp70 49fold, and also 11.5fold the activity of the isolated ATPase domain. Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides
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human Hsp70 escort protein
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human Hsp70 escort protein (Hep) possesses the unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins
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J-protein
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J-protein splice hTid-1L, larger isoform and hTid-1S, smaller isoform. At a morlar ratio of mtHsp70 to J-protein, a 3.2fold stimulation is observed with hTid-1L for wild-type protein. hTid-1S shows an 11fold stimulation for wild-type mtHp70
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KCCC
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comparable stimulation of ATPase activity of mtHsp70 and Dnak
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KKCC
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comparable stimulation of ATPase activity of mtHsp70 and Dnak
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Mdj1p
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mitochondrial co-chaperone stimulates ATPase activity of both mtHsp70 and Dnak 5-6fold, 20fold activation can be achieved by different molar ratios
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Mdj2
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stimulates ATPase activity of mtHsp70 to the same extent as TIM14, forms a complex with TIM16, which does not interfer with the TIM16-TIM14 complex and cannot support cell growth, but overexpression of Mdj2 fully restores growth of cells lacking TIM14
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Mge1p
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mitochondrial co-chaperone stimulates ATPase activity of both mtHsp70 and Dnak 5-6fold, 20fold activation can be achieved by different molar ratios
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NRLLLTG
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substrate binding allosterically stimulates ATPase activity of mtHsp70 2.5 fold, that of Dnak 5fold
Pam18
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stimulates mtHsp70's ATPase activity, Pam18 is less active in complex with Pam16, which is not sufficient to have significant effects on mitochondrial protein import or cell growth
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TIM14
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additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00068
ATP
Homo sapiens
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pH 7.5, 25°C, full-length mtHsp70 in absence of Hep
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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quantitative analysis of ATP binding activity of recombinant mtHsp70
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 76.2
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mtHsp70 binding kinetics are faster at higher temperatures
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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TIG-1 cell, overexpression of mortalin extends the in vitro lifespan of the fibroblasts
Manually annotated by BRENDA team
additional information
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NARF cell, stably transfected with isopropyl beta-D-thiogalactoside-inducible p14ARF
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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cpHsc70-1 and cpHsc70-2 are highly conserved proteins, imported into chloroplasts, analyzed by fluorescence microscopy
Manually annotated by BRENDA team
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YFP-fusion proteins of cpHsc70-1 and cpHsc70-2 are predominantly stromal, analyzed by fluorescence microscopy
Manually annotated by BRENDA team
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low level, analyzed by fluorescence microscopy
Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
44000
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SDS-PAGE, possibly the ATPase domain of mtHsp70
58000
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SDS-PAGE, fragment in the presence of ATP
81000
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1 * 81000, full-length enzyme in absence of ADP
86000
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1 * 86000, full-legth enzyme in presence of ADP, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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full-legth enzyme in presence of ADP, SDS-PAGE
monomer
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1 * 81000, full-length enzyme in absence of ADP; 1 * 86000, full-legth enzyme in presence of ADP, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
study of the structural interactions of Hsc70 ATPase domains with four different NEFs (nucleotide exchange factors) using PDB-ID: 1HPM. Two classes of key residues are distinguished in the domain: highly conserved residues involved in the nucleotide binding and not conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs, i.e. N57, R258, R262, E283, And D285. Functional variability accompanied by structural variability at the co-chaperone binding sites and conservation/robustness both in terms of sequence and structural dynamics at the nucleotide binding sites are evolutionary optimized and is essential for adapting to interactions with differnent cofactors while meintaining ATPase activity
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
51.6
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thermal denaturation of mtHsp70 shows three endotherms centered at 51.6, 67.5 and 76.2°C, the endotherm at 51.6°C may represent the unfolding of a more stable ATPase domain
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
mtHsp70 and Hsp70 ATPase domain remain soluble in the absence of Hep and nucleotides when stored in Tris at pH 8.0. In the case of the ATPase domain, the protein remains soluble at concentrations as high as 0.2 mM even when stored for 24 h at room temperature. However, the mtHsp70 ATPase domain could not be stably stored at high concentrations for a similar period of time in buffers having pH values more closely resembling the physiological environment within Escherichia coli where these proteins are overexpressed, unless it is incubated with equimolar Hep
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity purification
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by ion exchange, ATP-agarose affinity and hydroxyapatite chromatographies
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isolation of mitochondria from Saccharomyces cerevisiae cells recombinantly expressing His-tagged mtHsp70, and further purification by nickel affinity chromatography
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purified on nickel-Sepharose, by ion exchange on a Mono Q column, and by gel filtration
recombinant C-terminally His-tagged enzyme from Triticum aestivum germ lysates by nickel affinity chromatography
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recombinant His-tagged full-length enzyme and isolated ATPase domain from Escherichia coli by nickel affinity chromatography, ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
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recombinant His-tagged mtHsp70
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recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chimeric Hsp70s overexpressed in BB1553 cells, mtHsp70 overexpressed in the yeast strain YKN3B
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cloned in Escherichia coli, expression of ATPase and co-expression of the Hep1 in plasmids containing Ssc1 constructs
cpHsc70-1 and cpHsc70-2 cDNAs are PCR amplified and cloned into NdeI and XhoI sites of pET28a and introduced into Escherichia coli BL21(DE3)
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expression of mtHsp70 and its isolated ATPase domain, as well as the ATPase fused to the peptide-binding domain of HscA, in Escherichia coli as insoluble proteins; HEK-293 cells are transiently transfected with pHep-EGFP, expression of C-terminally His-tagged Hsp70 without its N-terminal mitochondrial targeting sequence in Escherichia coli, the enzyme is insoluble in absence of human escort protein Hep, also as isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone
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for purification of the His-tagged human mtHsp70, coexpression is carried out with yeast Hsp70 escort protein Hep in Escherichia coli BL21(DE3)
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recombinant production of the C-terminally His-tagged enzyme by Triticum aestivum germ lysates, co-expression with the proteins of the interactome of the TIM23 core complex
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SSC1 expression in Saccharomyces cerevisiae
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the sequence encoding human Hsc70 is inserted into the pProExHTa vector
the sequence encoding rat Hsc70 is inserted into the pET11a vector
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression patterns for cpHsc70-1 and cpHsc70-2 genes analyzed using the GUS (beta-gucuronidase) reporter gene. High Expression determined throughout seedlings, in all tissues of mature plants, unaltered in the drak or low light. Both genes are ubiquitously expressed at high levels, cpHsc70-1 slightly higher than cpHsc70-2, especially in roots
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y196A/N198A/D199A
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YND mutant, shows 3fold elevated basal activity in comparison with wild-type. The mutant also shows a significant decrease in Hsp70 escort protein Hep stimulation as in comparison to the wild-type protein. The YND mutant also exhibits a decreased stimulation of the J-protein splice variant hTid-1S in comparison of the wild-type further supporting the mutually exclusive nature of Hep and J-protein interaction at the ATPase domain of human mtHsp70
E240A/V241A
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the mutation is supposed to affect both a cluster of charge interactions with Lys546 and Arg437 of the mtHsp70 substrate-binding domain, as well as hydrophobic interactions at the interface between the two Hsp70 domains, e.g. Ala542 within alpha-helix A of the substrate-binding domain. Lethal mutation due to a defect in allosteric regulation, phenotype, overview. No complementation of a ssc1 null mutant
N175A/D176A
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the mutation abolishes a salt bridge that is formed between Asp176 of the nucleotide-binding domain and Lys544 of the substrate-binding domain. The mutant shows a temperature-sensitive phenotype with defect growth at 30°C due to a defect in allosteric regulation, phenotype, overview. No complementation of a ssc1 null mutant
Y173A
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residue Tyr173, that is exposed on the molecular surface and located close to both the nucleotide-binding site and the putative domain interface. The mutant displays normal growth that is indistinguishable from cells expressing wild-type Ssc1. Complementation of a ssc1 null mutant
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
additional information