Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate

Abstract

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of ؎35°with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.allostery ͉ dipolar couplings ͉ dynamics ͉ NMR ͉ structure H sp70 (heat shock 70 kDa) chaperone proteins are central to protein folding, refolding, and trafficking in organisms ranging from Archae to Homo sapiens, both at normal and at stressed conditions (for a review, see ref. 1). Recently, Hsp70s have been linked to breast and colon cancer (2) and to diseases such as Alzheimer's (3), Parkinson's (4), and Huntington's (5) diseases. In this report, DnaK, the canonical Hsp70 molecular chaperone protein from Escherichia coli, is studied. In the ADP state, DnaK, like other Hsp70s, binds to exposed hydrophobic residues of unfolded or partially misfolded proteins. Upon ATP binding, which induces an allosteric conformational change, DnaK releases the client protein (6). This process is tightly regulated by cochaperone proteins (7). DnaK consists of three subdomains. The structure of the nucleotide-binding domain (NBD, residues 1-370), was solved by crystallography (8). It competitively binds ATP and ADP and can slowly hydrolyze ATP (9). Structures for the 15-kDa substratebinding domain (SBD, residues 390-600) were solved in different forms by crystallography (10) and NMR (11-13). It harbors the hydrophobic substrate-binding cleft. Here, this subdomain is referred to as BETA. A subsequent 10-kDa subdomain of ␣-helical structure (residues 510-638) was characterized by NMR (14) and crystallography (15). This subdomain, referred to as the LID, plays a key role in regulating the kinetics of substrate binding (16,17).Recently, structures have become available comprising both the NBD and SBD. Our group has used NMR methods to determine the global 3D solution structure of an NBD-SBD construct (residues 1-501) of Thermus thermophilus DnaK (18). A crystal structure of Bos taurus Hsc70 (residues 1-554 and E213A/D214A) was reported (19). A crystal structure for Geobacillus kaustophilus DnaK (residues 1-509) was determined (20). Furthermore, a crystal structure of Saccharomyces cerevisiae Hsp110 (2-659), which is a Hsp70 ho...

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

“…2D). Direct support for these predictions is provided by the finding that in solution the portion of E. coli DnaK following residue 603 has been found to lack stable structure (6), is susceptible to proteolysis, and its removal facilitates crystallization of the SBD (29).…”

“…In the ⌬secB assay with controllable dnaK expression, colonies of E. coli strain GP502 transformed with pMS119-dnaK plasmids were used to inoculate overnight cultures grown at 30°C in LB media supplemented with 0.5% arabinose, 50 mg/liter ampicil- (6,29,58), the NBD (green) is joined to the SBD (blue) through a short interdomain linker (red). A peptide substrate is sandwiched between the SBD ␤-sandwich subdomain and ␣-helical lid, with 35 crystallographically unresolved residues in the C-terminal tail.…”

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

“…We therefore suggest a model accounting for the polypeptide unfoldase activity of Hsp110, in which the ␣-helical lid may depart from its stable position alongside the nucleotide-binding domain observed in the crystal structures and adopt during ATP hydrolysis an extensively opened position capable of binding to large bulky hydrophobic misfolded domains of a substrate polypeptide and, upon ATP hydrolysis, operate a large clamping movement toward the ␤-sheet base subdomain, as in the case of DnaK (57), that may in turn cause the unfolding of the clamped misfolded polypeptide segment (43).…”

Mapping the conformation of a client protein through the Hsp70 functional cycle

“…During the course of its function DnaK cycles through ADP-bound, ATP-bound, and NF states (2). In both NF and ADPbound conformations the NBD and SBD of DnaK are separated from each other, and the helical lid is closed, with small k on ,k off values and high affinities for substrate (2,6). In contrast, in the lowaffinity, fast k on ,k off ATP-DnaK state the two domains are docked H-15 N TROSY HSQC spectrum of hTRF1 bound to NF-DnaK and compared it to the corresponding spectrum of the ADP-DnaK bound state.…”