NMR STRUCTURE DETERMINATION OF THE ESCHERICHIA-COLI DNAJ MOLECULAR CHAPERONE - SECONDARY STRUCTURE AND BACKBONE FOLD OF THE N-TERMINAL REGION (RESIDUES-2-108) CONTAINING THE HIGHLY CONSERVED J-DOMAIN

DnaJ from Escherichia coli is a 376-amino acid protein that functions in conjunction with DnaK and GrpE as a chaperone machine. The N-terminal fragment of residues 2-108, DnaJ-(2-108), retains many of the activities of the full-length protein and contains a structural moth, the J domain Of residues 2-72, which is highly conserved in a superfamily of proteins. In this paper, NMR spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of DnaJ (2-108). By using C-13/N-15 doubly labeled DnaJ-(2-108), nearly complete sequence-specific assignments were obtained for H-1, N-15, C-13(alpha), and C-13(beta), and about 40% of the peripheral aliphatic carbon resonances were also assigned. Four cu-helices in polypeptide segments of residues 6-11, 18-31, 41-55, and 61-68 in the J domain were identified by sequential and medium-range nuclear Overhauser effects. For the J domain, the three-dimensional structure was calculated with the program DIANA from an input of 536 nuclear Overhauser effect upper-distance constraints and 52 spin-spin coupling constants. The polypeptide backbone fold is characterized by the formation of an antiparallel bundle of two long helices, residues 18-31 and 41-55, which is stabilized by a hydrophobic core of side chains that are highly conserved In homologous J domain sequences. The Gly/Phe-rich region from residues 77 to 108 is flexibly disordered in solution.