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 bendin g DNA. An x-ray structure has been determined previously [Tanaka et al . (1984 ) Nature, Vol. 310, pp. 376-381], but no structure could be es tablished for a lai ge parr of the supposed DNA-binding beta-arms. Dis tance 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 Angstrom for the well-defined region (residues 2-54 and 75-90). The structure of the core is very s imilar to that observed in the x-ray structure, with a pairwise RMSD o f 1.06 Angstrom. The structure of the beta-hairpin arm contains a doub le flip-over at the prolines in the two strands of the beta-arm. Heter onuclear N-15 relaxation measurements indicate that the beta-arm and t he tip of the beta-arm is flexible. This explains the disorder observe d in the solution and x-ray structures of the beta-arm with respect to the col-e of the protein. Overlayed onto itself the beta-arm is bette r defined, with an backbone RMSD of 1.0 Angstrom calculated for residu es 54-59 and 69-74. The tip of the arm adopts a well-defined 4 : 6 bet a-hairpin conformation. Changes in amide N-15 and H-1 chemical shifts upon titrating DNA al-e most pronounced for the residues in the beta-h airpin arm and for the residues in the second half of the third alpha- helix. Heteronuclear N-15 relaxation data for free ee and complexed HU Bst show that the arms become structured ripen DNA binding. Together w ith chemically induced nuclear polarization measurements on a mutant H UBst (M69Y; V76Y) this shows that the beta-hairpin arm is involved in direct DNA interaction. (C) 1997 John Wiley & Sons, Inc.