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 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.
Boelens
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R
Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Vis
H
H
Vorgias
C E
CE
Wilson
K S
KS
Kaptein
R
R
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
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C040330:histone-like protein HU, bacteria