H-1 AND N-15 MAGNETIC-RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE, AND TERTIARY FOLD OF ESCHERICHIA-COLI DNAJ(1-78)

We report the H-1 and N-15 chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly N-15-labeled DnaJ(1-78), which is a stable, folded fragment. Standard N-15-edited NMR techniques afforded complete assignment of the backbone amide H-1 and N-15 pairs and partial assignment of the side-chain H-1 and N-15 atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D N-15-separated and 2D homonuclear NOESY spectra as well as (3)J(HNH alpha), coupling constants obtained from a DQF-COSY spectrum and a N-15-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four alpha-helices and no beta-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.