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Título : The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain
Autor : Moro, Fernando; Fernández-Sáiz, Vanesa; Muga, Arturo
Palabras clave : DnaK
Fecha de publicación : feb-2006
Editor: Cold Spring Harbor Laboratory. Press
Citación : Protein Science 15(2): 223–233 (2006)
Resumen: 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 a-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 cagednucleotides. 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 a-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 (428C).
Versión del editor: http://dx.doi.org/10.1110/ps.051732706
URI : http://hdl.handle.net/10261/3535
DOI: 10.1110/ps.051732706
ISSN: 1469-896X
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