Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.