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 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 15N 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 15N and 1H 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 15N 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

NMR elucidation of monomer–dimer transition and conformational heterogeneity in histone‐like DNA binding protein of Helicobacter pylori

“…Significantly lower than the average T 1 values and higher than the average T 2 ‐values were seen for residues corresponding to antiparallel β ‐hairpin structure formed by β 3 and β 4 strands (Figure a,b ) , indicating fast (nanosecond to microsecond time scale) mobility and structural plasticity in β‐arms relative to the core. Have Consistent with previous reports, the flexible β‐arms relative to the core provide an exquisitely control to dimeric Hup to grasp DNA into the saddle domain, and thus rendering functional morphology to perform various DNA dependent cellular transactions.…”

HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

“…The residue corresponding to Thr 65 of HupB has been shown to be located in the bend of the ␤-ribbon arm in Bacillus stearothermophilus (46) and the residues corresponding to Thr 68 and Thr 74 in the return arm of the ␤-ribbon. Of the three residues, Thr 65 and Thr 74 showed conservation or conservative substitution to serine in HupB homologs in actinomycetes (see Fig.…”

β-Arm flexibility of HU fromStaphylococcus aureusdictates the DNA-binding and recognition mechanism

“…To understand the DNA-recognition mechanism of HU from S. aureus (SHU), the atomic structures of apo SHU and SHU-DNA must be determined. Several HU structures with and without DNA have been determined using X-ray crystallography and NMR (Tanaka et al, 1984;Boelens et al, 1996;Swinger et al, 2003;Christodoulou et al, 2003;White et al, 1999), in which the proteins usually exist as dimers. Structural studies of HU proteins in the absence of DNA and the crystal structure of the HU-DNA complex showed that the structures share a common fold that consists of a large -helical 'body' with two protruding -ribbon 'arms'.…”