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ATPase Domain (atpase + domain)

Distribution by Scientific Domains

Chemistry	50%

Selected Abstracts

The allosteric transition in DnaK probed by infrared difference spectroscopy.

PROTEIN SCIENCE, Issue 2 2006
Concerted ATP-induced rearrangement of the substrate binding domain
SBD, substrate binding domain; TR-IR, time resolved infrared spectroscopy; IR, infrared spectroscopy Abstract The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal ,-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the ,-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42°C). [source]

In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain

THE PLANT JOURNAL, Issue 6 2007
Christopher James Snowden
Summary The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis. [source]

cDNA cloning and expression analysis of the myosin heavy chain (MYH) gene of the mandarin fish Siniperca kneri

AQUACULTURE RESEARCH, Issue 4 2009
Jianshe Zhang
Abstract In this study, we applied RT-PCR and cDNA cloning techniques to clone myosin heavy chain (MYH) cDNA from muscle tissues of the mandarin fish Siniperca kneri. The cDNA was determined to be of 6987 base pairs in length, encoding a peptide of 1937 amino acids (Genbank accession no. EF446616). A search of encoded protein sequences in the NCBI conserved domain database indicated the presence of all known protein domains for MYH proteins, i.e. the myosin motor domain in the N-terminal region, the DIL domain at the C-terminus, and the ATPase domain. The MYH gene and its protein were expressed predominantly in muscle tissues and weakly in cardiac tissues. Developmentally, the MYH gene was first expressed in the muscle formation stage and continued later on. Our work provided a novel mypsin heavy chain gene sequence in fish biology and the results indicate that the MYH gene and the protein it encodes are important for the growth and development of the mandarin fish, as well as its muscle characterization. [source]

Crystallization of the 43,kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocin

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002
V. Lamour
The 43,kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43,kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10,µm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3,Å allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin. [source]

X-ray structure determination at low resolution

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2009
Axel T. Brunger
As an example of structure determination in the 3.5,4.5,Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3,Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at ,4,Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible. [source]