¹H and ¹⁵N Magnetic Resonance Assignments, Secondary Structure, and Tertiary Fold of Escherichia coli DnaJ(1-78)

We report the ¹H and ¹⁵N 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 ¹⁵N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard ¹⁵N-edited NMR techniques afforded complete assignment of the backbone amide ¹H and ¹⁵N pairs and partial assignment of the side-chain and ¹⁵N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D ¹⁵N-separated and 2D homonuclear NOESY spectra as well as ³JHNHCX coupling constants obtained from a DQF-COSY spectrum and a ¹⁵N-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 a-helices and no β-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 at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.