title: Structure and dynamics of the DNA binding protein HU from Bacillus stearothermophilus by NMR spectroscopy.
abstract: The DNA-binding protein HU from Bacillus stearothermophilus (HUBst) is a dimer with a molecular weight of 195 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 beta-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 A 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 A. The structure of the beta-hairpin arm contains a double flip-over at the prolines in the two strands of the beta-arm. Heteronuclear 15N relaxation measurements indicate that the beta-arm and the tip of the beta-arm is flexible. This explains the disorder observed in the solution and x-ray structures of the beta-arm with respect to the core of the protein. Overlayed onto itself the beta-arm is better defined, with a backbone RMSD of 1.0 A calculated for residues 54-59 and 69-74. The tip of the arm adopts a well-defined 4 : 6 beta-hairpin conformation. Changes in amide 15N and 1H chemical shifts upon titrating DNA are most pronounced for the residues in the beta-hairpin arm and for the residues in the second half of the third alpha-helix. Heteronuclear 15N relaxation data for free and complexed HUBst show that 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 beta-hairpin arm is involved in direct DNA interaction.
authors.affiliation: Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Boelens
authors.forename: H
authors.initials: H
authors.lastname: Vis
authors.forename: C E
authors.initials: CE
authors.lastname: Vorgias
authors.forename: K S
authors.initials: KS
authors.lastname: Wilson
authors.forename: R
authors.initials: R
authors.lastname: Kaptein
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C040330:histone-like protein HU, bacteria