Nucleotide Binding to the Heat‐Shock Protein DnaK as Studied by ESR Spectroscopy

Neuhofen, Sonja; Theyssen, Holger; Reinstein, Jochen; Trommer, Wolfgang E.; Vogel, Pia D.

doi:10.1111/j.1432-1033.1996.0078h.x

Cited by 14 publications

(6 citation statements)

References 20 publications

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“…This is indicative of the formation of a ternary DnaK‚GrpE‚MABA-ADP complex at high GrpE concentrations, meaning that there is residual affinity of MABA-ADP for the DnaK‚GrpE complex. The formation of a ternary complex of DnaK, GrpE, and a spinlabeled nucleotide analog was observed previously by ESR spectroscopy (Neuhofen et al, 1996).…”

Section: Resultssupporting

confidence: 70%

GrpE Accelerates Nucleotide Exchange of the Molecular Chaperone DnaK with an Associative Displacement Mechanism

et al. 1997

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The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.

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Section: Resultssupporting

confidence: 70%

GrpE Accelerates Nucleotide Exchange of the Molecular Chaperone DnaK with an Associative Displacement Mechanism

et al. 1997

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“…Spin-labeled nucleotides have been used successfully to study nucleotide binding to the nucleotide binding sites of F 1 -ATPases from different species (26-31, for review see ref 32) as well as chaperones GroEL, DnaK, and human Hsp90 (33)(34)(35), the G-protein, p21ras (36), and the calcium channel, RyR1 (Dias, J. M. et al, manuscript in preparation).…”

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confidence: 99%

Nucleotide Binding to the Multidrug Resistance P-Glycoprotein as Studied by ESR Spectroscopy

et al. 2005

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Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.

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“…GrpE function is analogous to that of other well-known nucleotide exchange factors, such as the elongation factor EFTs and RCC1, which are GEFs for the small GTPases EFTu and Ran, respectively, with many similarities with respect to their kinetics. For EFTs and RCC1, as well as for GrpE, transient ternary complexes of exchange factornucleotide hydrolase-nucleotide diphosphate can be detected (Klebe et al 1995;Neuhofen et al 1996;Packschies et al 1997;Gromadski et al 2002). Thermodynamic balance requires that the nucleotide exchange factors and the nucleotide mutually reduce affinity for the nucleotide hydrolase, and this has been experimentally confirmed: GrpE reduces the affinity of DnaK for ADP by 200-fold, and ADP reduces the affinity of GrpE for DnaK by 200fold (Packschies et al 1997).…”

Section: Kinetics Of Nucleotide Exchangementioning

confidence: 91%

GrpE, a nucleotide exchange factor for DnaK

Harrison

2003

Cell Stress Chaper

110

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The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.

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Nucleotide Binding to the Heat‐Shock Protein DnaK as Studied by ESR Spectroscopy

Cited by 14 publications

References 20 publications

GrpE Accelerates Nucleotide Exchange of the Molecular Chaperone DnaK with an Associative Displacement Mechanism

GrpE Accelerates Nucleotide Exchange of the Molecular Chaperone DnaK with an Associative Displacement Mechanism

Nucleotide Binding to the Multidrug Resistance P-Glycoprotein as Studied by ESR Spectroscopy

GrpE, a nucleotide exchange factor for DnaK

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