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J. Biol. Chem., Vol. 279, Issue 19, 19600-19606, May 7, 2004

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The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site^*

Fernando Moro, Vanesa Fernández-Sáiz, and Arturo Muga¶

From the Unidad de Biofísica and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, 48080 Bilbao, Spain

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25 °C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37 °C) and stress (42 °C) temperatures, removal of the N-terminal half of B and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and -sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.

Received for publication, January 28, 2004 , and in revised form, February 25, 2004.

^* This work was supported by Universidad del País Vasco Grant UPV 13505/2001 and Ministerio de Ciencia y Tecnología Grant MCYT BMC 2001/0950. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a postdoctoral fellowship funded by the Basque Government.

Recipient of a predoctoral fellowship funded by the Basque Government.

^¶ To whom correspondence should be addressed. Tel.: 349-4610-2624; Fax: 349-4464-8500; E-mail: gbpmuvia{at}lg.ehu.es.

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