Structure and dynamics of the DNA binding protein HU from Bacillus stearothermophilus by NMR spectroscopy

The DNA‐binding protein HU from Bacillus stearothermophilus (HUBst) is a dimer with a molecular weight of 19.5 kDa that is capable of bending DNA. An x‐ray structure has been determined previously [Tanaka et al. (1984) Nature, Vol. 310, pp. 376–381], but no structure could be established for a large part of the supposed DNA‐binding β‐arms. Distance geometry and restrained molecular dynamics using nmr restraints were used to generate a set of 25 structures. These structures display a backbone rms deviation (RMSD) of 0.36 Å for the well‐defined region (residues 2–54 and 75–90). The structure of the core is very similar to that observed in the x‐ray structure, with a pairwise RMSD of 1.06 Å. The structure of the β‐hairpin arm contains a double flip‐over at the prolines in the two strands of the β‐arm. Heteronuclear 15 N relaxation measurements indicate that the β‐arm and the tip of the β‐arm is flexible. This explains the disorder observed in the solution and x‐ray structures of the β‐arm with respect to the core of the protein. Overlayed onto itself the β‐arm is better defined, with an backbone RMSD of 1.0 Å calculated for residues 54–59 and 69–74. The tip of the arm adopts a well‐defined 4 : 6 β‐hairpin conformation. Changes in amide 15 N and 1 H chemical shifts upon titrating DNA are most pronounced for the residues in the β‐hairpin arm and for the residues in the second half of the third α‐helix. Heteronuclear 15 N relaxation data for free and complexed HUBst show that the arms become structured upon DNA binding. Together with chemically induced nuclear polarization measurements on a mutant HUBst (M69Y; V76Y) this shows that the β‐hairpin arm is involved in direct DNA interaction. © 1997 John Wiley & Sons, Inc. Biopoly 40: 553–559, 1996