Spiral structure of Escherichia coli HUalphabeta provides foundation for DNA supercoiling

Abstract

We determined the crystal structure of the Escherichia coli nucleoid-associated HUalphabeta protein by x-ray diffraction and observed that the heterodimers form multimers with octameric units in three potential arrangements, which may serve specialized roles in different DNA transaction reactions. It is of special importance that one of the structures forms spiral filaments with left-handed rotations. A negatively superhelical DNA can be modeled to wrap around this left-handed HUalphabeta multimer. Whereas the wild-type HU generated negative DNA supercoiling in vitro, an engineered heterodimer with an altered amino acid residue critical for the formation of the left-handed spiral protein in the crystal was defective in the process, thus providing the structural explanation for the classical property of HU to restrain negative supercoils in DNA.

Fig. 1.

Snapshots of electron density maps of residues 12 and 13 as finger prints of the α and β chains. (a) E12 and K13 of the α-subunit. (b) A12 and G13 of β. (c) The same region of a with E12 and K13 omitted. The 2F_o − F_c map cutoff at 1 is in green. The positive F_o − F_c map cutoff at 3 is in red. The red and blue text indicates separate HU dimer molecules.

Fig. 2.

Residues that contribute to the hydrophobic core of E. coli HU. Those in red directly contribute to dimerization. The underlined residues are different in α and β chains. The italicized residues are those whose counterparts in the opposite chain are polar or charged.

Fig. 3.

Top views of crystal packing: an octameric structure with three possible arrangements. (a) The crystal packing lattice. (b) Arrangement I octamer. (c) Arrangement II octameric unit of a right-handed spiral multimer. (d) Arrangement III octameric unit of a left-handed spiral multimer. Red and blue represent the α- and β-subunits, respectively.

Fig. 4.

Diagrams of the interdimeric interfaces of E. coli HUαβ protein. (a) Interface “a” involves interactions between residues (E12 and E15-L16-S17-K18-T19) of two adjacent symmetry-related α-subunits located in the turn between helix 1 and helix 2. (b) Interface “b” shows extensive interactions involving a Ni-ion, a Cl-ion, three H₂O molecules, α-M1, α-K3, α-T4, α-Q5, and α-D40 from one dimer, and α-E38, α-H54, and β-D40 from the adjacent dimer. (c) Interface “c” involves the side chain of the β-E38, which rotates to form two H-bonds with the backbone amines of α-G46 and α-K83, respectively, from the interacting adjacent dimer. Red and blue represent segments of the subunits of α and β, respectively.

Fig. 5.

Stereoviews of modeled HUαβ octameric unit of the left-handed multimer with DNA fragment as a repeating unit of a spiral structure. The α-subunit is in red, and the β-subunit is in blue. (a) Top view. (b) Side view. The HUαβ multimeric spiral is wrapped by DNA in a left-handed solenoidal structure.

Fig. 6.

Two-dimensional agarose gel electrophoretic patterns of circular DNA plasmids. Five nanomolar relaxed plasmid samples were treated with wild-type HUαβ (a) and HUα⁺β-E38A (b) at 0, 1.79, 2.08, 2.50, 3.13, 4.17, and 6.25 μM concentrations (lanes 1–7, respectively) in the presence of topoisomerase I (see text).

Fig. 7.

EMSA of HU-DNA. Five nanomolar relaxed plasmid samples were treated with wild-type α⁺β⁺ (a) or mutant α⁺β-E38A (b) HU at 0, 1.25, 1.39, 1.56, 1.79, 2.08, 2.50, 3.13, 4.17, 6.25, 12.5, 25.0, and 50 μM concentrations (lanes 1–13, respectively).