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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...

NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.

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