Importance of the D and E Helices of the Molecular Chaperone DnaK for ATP Binding and Substrate Release ² Sergey V. Slepenkov, ‡ Brandi Patchen, ‡ Kenneth M. Peterson, § and Stephan N. Witt* ,‡ Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Louisiana State UniVersity Health Sciences Center, 1501 Kings Highway, ShreVeport, Louisiana 71130-3932 ReceiVed January 22, 2003; ReVised Manuscript ReceiVed March 26, 2003 ABSTRACT: The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five R-helices (RA-RE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the RD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k off equal to 7.6 and 8.9 s -1 , respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k off jumps to 207 s -1 . (ii) The presence of the RB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s -1 , whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s -1 . Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the RE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain. The Escherichia coli 70-kDa molecular chaperone DnaK folds, transports, and assembles other proteins in an ATP- dependent activity cycle that is regulated by the cochaper- ones, GrpE and DnaJ (1-3). DnaK cycles from an ADP- bound, high-affinity state that tightly binds unfolded substrate to an ATP-bound, low-affinity state that weakly binds substrate. GrpE is the nucleotide exchange factor that catalyzes the release of ADP from ADP-DnaK complexes (4-6). ADP release in turn permits ATP binding which induces the high-to-low affinity transition. DnaJ promotes the reverse transition (7, 8). DnaK is composed of three domains: the ATPase domain, the substrate-binding domain, and the lid comprise residues 1-388, 389-508, and 509-638, respectively. The ATPase domain is a bilobed structure that contains a deep channel between the two lobes (9, 10); nucleotide binds at the base of the channel. The substrate-binding domain consists of a uniquely folded -sandwich subdomain followed by an R-helical domain that consists of five antiparallel R-helices (Figure 1A) (11, 12). This R-helical domain is like a lid over the -sandwich subdomain (12). A network of hydrogen bonds and a salt bridge links the lid noncovalently to the -sandwich. The bound peptide interacts with the -sandwich but not the lid (12). The role of the lid in the chaperone activity cycle is still not precisely understood. Interdomain coupling occurs in the absence of the lid (13, 14), although ATP-induced peptide dissociation is significantly accelerated (15) compared to the wild-type protein. The 33 C-terminal residues of DnaK, which constitutes a flexible, mobile region of the protein, has no known function (16). There are intriguing hints that the lid region of DnaK and other Hsp70s interacts with DnaJ (17). Recent experiments have shown that deletion of the bulk of DnaK’s lid, residues 518-638, increases the rate of ATP- induced peptide release from 7.6 to 299 s -1 (18). Intrigued by this finding, and to determine the relative importance of each helix to peptide release, we have undertaken a study in which the five helices that constitute DnaK’s lid plus the 30-residue flexible tail were sequentially deleted. Each variant was then tested for a variety of activities, viz., ATP- induced peptide dissociation, ATP hydrolysis, and the ATP- induced high-to-low affinity conformational change. We show that the RE and RD helices are key helices for lid stability. The RD helix is the key helix with respect to peptide release because when this helix is deleted the rate constant for ATP-induced peptide release jumps from 7.6 to 207 s -1 . A model is proposed for how RD stabilizes the lid and creates long-lived DnaK-peptide complexes. ² This work was supported by a grant from the National Institutes of Health (GM 51521) (to S.N.W.). * To whom correspondence should be addressed. Tel: (318) 675- 7891. FAX: (318) 675-5180. E-mail: switt1@ lsuhsc.edu. ‡ Departments of Biochemistry and Molecular Biology. § Microbiology and Immunology. 5867 Biochemistry 2003, 42, 5867-5876 10.1021/bi034126v CCC: $25.00 © 2003 American Chemical Society Published on Web 04/23/2003