Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment.

Flaherty, Kevin; Wilbanks, Sigurd M.; DeLuca-Flaherty, Camille; McKay, David

doi:10.1016/s0021-9258(18)99961-8

Cited by 143 publications

(70 citation statements)

References 19 publications

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“…The bidentate complex is stabilized by water molecules, which are coordinated by D 10 , K 71 , E 175 , and D 199 (Figure 3A). 60 The location of K 71 within the active site is also maintained across the various structures (Figure 3A); K 71 is proposed to facilitate the nucleophilic attack on ATP via positioning a water molecule in line with the γ-phosphate. 61 The prehydrolysis state of GRP78, visualized with GRP78 bound to the nonhydrolyzable analogue Mg 2+ •AppCHp (PDB entry 5F2R) (Figure 3B), 59 shows also the same active site configuration we observe for the Hsc70 Mg 2+ •ADP and P i structure (Figure 3A), reinforcing the high degree of active site conservation between Hsp70 members.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Focusing initially on our wild-type Mg 2+ ·ADP and P i structure, we observed a strong correlation of active site geometries with previously published Hsp70 structures. ,− A magnesium ion forms a bidentate complex with the β-phosphate of ADP and the hydrolyzed γ-phosphate. The bidentate complex is stabilized by water molecules, which are coordinated by D 10 , K 71 , E 175 , and D 199 (Figure A) . The location of K 71 within the active site is also maintained across the various structures (Figure A); K 71 is proposed to facilitate the nucleophilic attack on ATP via positioning a water molecule in line with the γ-phosphate .…”

Section: Resultsmentioning

confidence: 99%

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Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

et al. 2018

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The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.

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Section: ■ Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

et al. 2018

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“…Moreover, when triskelia rather than baskets were incubated in buffer C with hsc70 in the presence of ADP, under otherwise the same experimental conditions, no binding of hsc70 to clathrin was observed (data not shown). The existence of a stable hsC70ADP+Pi complex had been previously inferred from the analysis of the crystal structure of the nucleotide binding domain of hsc70 (12). More recently, Buxbaum and Woodman also had presented evidence for the longevity of the hsC70ADP÷Pi complex by demonstrating that the release of Pi from intact hsc70 is slow but enhanced by the presence of coated vesicles, especially by those purified from brain tissue (5).…”

Section: Discussionmentioning

confidence: 98%

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin.

Holstein

Ungewickell

1996

The Journal of Cell Biology

120

127

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Abstract. Auxilin was recently identified as cofactor for hsc70 in the uncoating of clathrin-coated vesicles (Ungewickell, E., H. Ungewickell, S.E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L.E.Greene, and E. Eisenberg. 1995. Nature (Lond.). 378: 632--635). By constructing different glutathione-S-transferase (GST)-auxilin fragments, we show here that cooperation of auxilin's J domain (segment 813-910) with an adjoining clathrin binding domain (segment 547-814) suffices to dissociate clathrin baskets in the presence of hsc70 and ATP. When the two domains are expressed as separate GST fusion proteins, the cofactor activity is lost, even though both retain their respective functions. The clathrin binding domain binds to triskelia like intact auxilin with a maximum stoichiometry of 3 and concomitantly promotes their assembly into regular baskets. A fragment containing auxilin's J domain associates in an ATP-dependent reaction with hsc70 to form a complex with a half-life of 8 min at 25°C. When the clathrin binding domain and the J domain are recombined via dimerization of their GST moieties, cofactor activity is partially recovered. The interaction between auxilin's J domain and hsc70 causes rapid hydrolysis of bound ATP. Release of inorganic phosphate appears to be correlated with the disintegration of the complex between auxilin's J domain and hsc70. We infer that the metastable complex composed of auxilin, hsc70, ADP, and Pi contains an activated form of hsc70, primed to engage clathrin that is brought into apposition with it by the DnaJ homologue auxilin. C LATHRIN-COATED vesicles are agents for the vectorial transport of selected membrane proteins from the plasma membrane and the trans-Golgi network to the endosomal system (4, 17). After budding and pinching off from the donor membrane, coated vesicles rapidly shed their coats. An enzyme catalyzing the uncoating of purified bovine brain coated vesicles was first purified from brain cytosol (34) and shown to be a member of the 70-kD heat shock protein (hsp) 1 70 family (7, 40). These proteins participate in numerous functions including the folding of newly synthesized proteins, translocation of proteins across membranes, disassembly of oligomeric complexes, and protection of proteins against irreversible denaturation under conditions of stress (16,19 as cofactor, the eukaryotic DnaJ homologue auxilin (41). Auxilin was originally identified as a clathrin assembly protein (2). This loose term refers to proteins that bind to clathrin and promote its polymerization into baskets. Auxilin binds to the region in the clathrin heavy chain that forms the hub and the proximal part of the triskelion leg. Light chains are not required for the interaction of auxilin with clathrin (25). Auxilin has in common with members of the DnaJ protein family a so-called J domain. This element of ~70 residues occupies the carboxyl-terminal end of the auxilin chain. When compared with the known J regions, it shows least homology to the archetypal J domain of bacterial DnaJ....

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“…Structural understanding of the Hsp70 mechanisms has been steadily advancing in recent years, evolving from the initial structural studies of the isolated domains to high-resolution structures of the full-length two-domain constructs and complexes with the nucleotides and small molecule inhibitors. Crystal structures and nuclear magnetic resonance (NMR) studies of the NBD constructs − and SBD complexes with the peptide substrates − have provided the first important insights into the structural basis of substrate specificity with Hsp70. Amide hydrogen exchange and mass spectrometry (HX-MS) studies of the two-domain DnaK constructs − along with subsequent biophysical experiments , have shown that the NBD and SBD units are largely independent in a domain-undocked form of the ADP-bound and nucleotide-free states, whereas ATP binding could stabilize a domain-docked structure that facilitates the interdomain signaling.…”

Section: Introductionmentioning

confidence: 99%

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks

Stetz

Verkhivker

2016

J. Chem. Inf. Model.

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Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

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Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment.

Cited by 143 publications

References 19 publications

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin.

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks

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