Thermus Thermophilus Dnak Nucleotide binding domain

This construct contains residues 1-381 of the deposited sequence with a 20
residue N-terminal leader sequence: MGSSHHHHHHSSGLVPRGSH

[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxy-oxolan-2-yl]methyl phosphono hydrogen phosphate

TROSY-driven NMR backbone assignments of the 381-residue nucleotide-binding domain of the Thermus Thermophilus DnaK molecular chaperone.

Letter to the Editor: TROSY-driven NMR backbone assignments of the 381-residue
nucleotide-binding domain of the Thermus Thermophilus DnaK molecular chaperone.
Revington, M., and Zuiderweg, E.R.P. J. Biol. NMR in press

We present an NMR investigation of the nucleotide-dependent conformational
properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein.
Conformational changes driven by ATP binding and hydrolysis in the N-terminal
NBD are believed to allosterically regulate substrate affinity in the
C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs
in different nucleotide states have, however, not shown significant structural
differences. We have previously reported the NMR assignments of the backbone
resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus
DnaK in the ADP-bound state. In this study we show, by assigning the NBD with
the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the
ATP-bound state. Chemical shift difference mapping of the two nucleotide states
identified differences in a cluster of residues at the interface between
subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP
state exhibited a single conformation, whereas the ADP state was in slow
conformational exchange between a form similar to the ATP state and another
state unique to the ADP-bound form. A model is proposed of the allosteric
mechanism based on the nucleotide state altering the balance of a dynamic
equilibrium between the open and closed states. The observed chemical shift
perturbations were concentrated in an area close to a previously described
J-domain binding channel, confirming the importance of that region in the
allosteric mechanism.

NMR study of nucleotide-induced changes in the nucleotide binding domain
of Thermus Thermophilus Hsp70 chaperone DnaK: Implications for the
allosteric mechanism.
Revington M, Holder TM, Zuiderweg ER.
J Biol Chem. 2004 Jun 2 [Epub ahead of print]

NMR study of nucleotide-induced changes in the nucleotide binding domain of Thermus thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism.

ASP44 converted to Isoasp when sample pH was
decreased from pH 7.4 to pH 6.0

DnaK nucleotide binding domain, Dnak NBD

DnaK homologue [Thermus thermophilus HB8]

chaperone protein DnaK (heat shock protein 70) [Thermus thermophilus HB8]

DnaK-homologue [Thermus thermophilus HB8]

chaperone protein dnaK [Thermus thermophilus HB27]

Chaperone protein dnaK [Thermus thermophilus SG0.5JP17-16]

chaperone protein DnaK [Thermus thermophilus JL-18]

chaperone protein DnaK [Thermus thermophilus]

RecName: Full=Chaperone protein DnaK; AltName: Full=HSP70; AltName: Full=Heat shock 70 kDa protein; AltName: Full=Heat shock pr

Backbone 1H, 13C, and 15N Chemical Shift assignments for the Nucleotide-binding
domain of Thermus Thermophilus DnaK

Samples were equilibrated at 55C for 20 minutes before data collection.