Conformational change of chaperone Hsc70 upon binding to a decapeptide: A circular dichroism study

Park, Kyusung; Flynn, Gregory C.; Rothman, James E.; Fasman, Gerald D.

doi:10.1002/pro.5560020304

Cited by 29 publications

(14 citation statements)

References 37 publications

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“…Circular dichroism spectroscopy analysis shows a significant amount of secondary structure: 35-40% ␣-helix, 40 -45% ␤-sheet, 5% ␤-turn, and 8 -12% random-coil as determined with SELCON software (48). These results are similar to those reported for Hsc70 (49,50). To address whether the N-terminal FLAG extension and the presence of an enterokinase site at the junction between the catalytic domain and the peptide binding domain would affect the catalytic properties of BiP, we engineered the plasmids pHis-BiP.ent and pHis-BiP and characterized the catalytic properties of purified recombinant HisBiP.ent and His-BiP proteins.…”

Section: Resultssupporting

confidence: 86%

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

Chevalier¹,

King²,

Wang³

et al. 1998

Journal of Biological Chemistry

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To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N-and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration-and temperaturedependent equilibrium. Heat shock proteins (HSPs) 1 are ubiquitous proteins found in all organisms and cell compartments. They are involved in cellular functions as varied as protein synthesis and proteolysis (1), stress tolerance (2), protein translocation into mitochondria (3) or the endoplasmic reticulum (4), and folding and assembly of proteins (5). Members of the HSP70 family are molecular chaperones that hold in common the ability to discriminate between unfolded polypeptide chains and native proteins. They participate in protein assembly by preventing aggregation due to hydrophobic interactions (6). HSP70 family members are highly conserved proteins, and share a structural organization in three domains: a 44-kDa N-terminal ATPase domain (7-9), an 18-kDa C-terminal peptide-binding domain (10, 11), and a less conserved C-terminal tail whose function and complete three-dimensional structure are unknown. The ATPase activity of HSP70 proteins is enhanced upon binding to synthetic peptides (12, 13). The size and nature of peptidic substrates required to fully stimulate the ATPase activity of HSP70 proteins have been determined to be 7-residue-long peptides, rich in aliphatic amino acids (14 -16). Detailed enzymological studies on monomeric, recombinant bovine Hsc70 revealed that binding of peptides increases the rate of ADP and inorganic phosphate release leading to an increase in the rate of ATP hydrolysis (17). In the same study, Takeda and McKay observed that the Mg.ADP.Hsc70 form has higher affinity for peptide than the Mg.ATP.Hsc70 form, in agreement with studies on Escherichia coli DnaK (18,19) and bovine brain Hsc70 (20). It was recently demonstrated that ATP-bound HSP70 proteins have high on/off rates of binding/release of substrates, whereas ADP-bound chaperones exhibit higher affinity for peptidic substrates through slower off rates (reviewed in Ref. 21). Many HSP70 proteins are also regulated by accessory proteins. A well studied system is the E. coli DnaK chaperone, which is regulated by the nucleotide exchange factor GrpE, which increases the rate of ADP/ATP exchange (22, 23) and by the highly conserved DnaJ, which increases the rate of ␥-phosphate cleavage (16,22). In addition to the different conformations HSP70 proteins adopt in the presence of substrates, they also self-associate into multiple oligomeric species that interconvert with each other (24 -26). In the bacterial system, addition of the dimeric cofactor GrpE to heterogeneous multimeric DnaK results in a slow conversion of oligomers into monomers and stabilization of (GrpE) 2 ⅐DnaK complexes (25). Mammalian Hs...

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Section: Resultssupporting

confidence: 86%

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

Chevalier¹,

King²,

Wang³

et al. 1998

Journal of Biological Chemistry

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“…Thus, our working hypothesis is that full activation of inactive protonated PhK (H-E) first requires a conformational change that results in a less negative surface charge before the obligatory Ca 2+ is able to induce an increase in the u 222 /u 208 ratio, which is a manifestation of the most active conformation, i.e., that subject to the least quaternary constraint by the inhibitory a and b subunits. Considering activation by [pH within the framework of this working hypothesis, we suggest that key residue(s) of the regulatory a and/or b subunits become deprotonated, causing a conformational change resulting in a less negative surface charge on the PhK complex, and that the binding of Ca 2+ to the d subunits of this deprotonated conformer indirectly decreases interactions between ahelices and b-sheets within PhK (i.e., increased u 222 /u 208 ratio) as part of the process of forming a more active conformer (Levitt and Chothia 1976;Chothia et al 1977;Arnold et al 1992;Park et al 1993). We assume that the decreased interactions of sheets and helices largely involve the a and b subunits.…”

Section: +mentioning

confidence: 96%

“…Because the ratio of negative ellipticity at 222 nm to 208 nm is an index of the extent of interaction between ahelices and b-sheets (Levitt and Chothia 1976;Chothia et al 1977;Manavalan and Johnson 1983;Arnold et al 1992;Park et al 1993), more detailed studies were carried out to investigate pH-and Ca…”

Section: Secondary Structure Characteristicsmentioning

confidence: 99%

Physicochemical changes in phosphorylase kinase associated with its activation

2008

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Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca 2+ -dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as [pH), but Ca 2+ dependence is retained. Little is known about the structural changes associated with PhK's activation by [pH and Ca 2+ , but activation by both mechanisms is mediated through regulatory subunits of the (abgd) 4 PhK complex. In this study, changes in the structure of PhK induced by [pH and Ca 2+ were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca 2+ and [pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH ;7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 À Ca 2+ ) versus that where it would be most active (pH 8.2 + Ca 2+ ), the joint activation by [pH and Ca 2+ is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of [pH and Ca 2+ in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.Keywords: phosphorylase kinase; Ca 2+ ; Tyr environment; secondary structure; tertiary structure; surface electrostatics; zeta potential Phosphorylase kinase (PhK), a complex regulatory enzyme in the cascade activation of glycogenolysis, is the only known kinase to phosphorylate and activate glycogen phosphorylase. By far, the most is known about the PhK from skeletal muscle (for reviews, see Heilmeyer Jr. 1991; Brushia and Walsh 1999), and that is the subject of this study. This PhK is a 1.3-MDa hexadecameric complex composed of four copies each of a single catalytic protein kinase subunit (g, 44.7 kDa) and three different regulatory subunits (a, 138.4 kDa; b, 125.2 kDa; and d, 17.7 kDa). Thus, its regulatory subunits account for 86% of the mass of the (abgd) 4 PhK complex. The a and b subunits are homologous and inhibitory, whereas the d subunits, which are endogenous molecules of calmodulin, are stimulatory. It is through allosteric sites on these regulatory subunits that PhK integrates metabolic (ADP), hormonal (cAMP and Ca 2+ ), and neural (Ca 2+ ) signals to control the rate of glycogen breakdown through large increases in the kinase activity of its catalytic g subunits. Ca 2+ ions presumably exert their stimulatory effect through binding to the d subunits, which in muscle couples contraction with energy production to supply

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“…Polypeptide substrates, such as reduced carboxymethylated lactalbumin (27,29) and a random library of decapeptides (30), were shown to alter the overall conformation of Hsp70s. Recently, peptide C, a peptide that binds to many Hsp70s, was shown to change the ATP-dependent conformation of DnaK to one resembling the ADP conformation (26).…”

Section: Hsp70mentioning

confidence: 99%

Conformations of the Nucleotide and Polypeptide Binding Domains of a Cytosolic Hsp70 Molecular Chaperone Are Coupled

Fung

Hilgenberg

Wang

et al. 1996

Journal of Biological Chemistry

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70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases that participate in protein folding by regulating protein-protein interactions. ATP binds to the highly conserved amino-terminal domain, whereas polypeptides bind to the less conserved carboxyl-terminal domain. These domains are functionally coupled. Polypeptides were previously shown to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity. We probed the structure of the yeast cytosolic Hsp70 Ssa1p using limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled. Ssa1p adopted three distinct conformations, nucleotide-free, ADP-dependent, and ATP-dependent. Complete conformational changes required K ؉ and Mg 2؉. Using aminoterminal sequencing, ATP-agarose chromatography, and a carboxyl-terminal-specific antibody, we mapped the locations of the major proteolytic fragments. Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains. Similarly, a polypeptide altered the conformations of both domains. These results indicate that the conformations of the nucleotide and polypeptide binding domains are coupled.

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Conformational change of chaperone Hsc70 upon binding to a decapeptide: A circular dichroism study

Cited by 29 publications

References 37 publications

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

Physicochemical changes in phosphorylase kinase associated with its activation

Conformations of the Nucleotide and Polypeptide Binding Domains of a Cytosolic Hsp70 Molecular Chaperone Are Coupled

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