H-NS forms a superhelical protein scaffold for DNA condensation

Abstract

The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS.

Fig. 1.

Structural basis for H-NS oligomerization. A, An oligomer of three symmetry-related (N-terminal oligomerization domain) dimers (Site 1), connected through their secondary dimer interface (Site 2). One dimer has been highlighted in color. B, Detailed view of dimerization Site 2. One protomer is displayed as secondary structure and stick model, the other as molecular surface. The surface is colored to highlight different properties of residues: blue, positively charged atoms; red, negatively charged atoms; green, hydrophobic atoms; salmon, polar oxygens; marine, polar nitrogens; yellow, sulfur. C, Amino acid sequences of H-NS from E. coli and S. typhymurium. The sequences are also shown for the H-NS paralogue StpA from S. typhymurium and the H-NS-like VicH from V. cholerae. Secondary structural features and residue numbers are included for reference.

Fig. 2.

Superhelical structure of H-NS in crystal lattice. A and B, 90° views of the superhelix formed by a chain of head-to-head and tail-to-tail linked H-NS molecules. Orientation of the molecules is taken from the crystal lattice. R15, R19, and K32 forming the positively charged, DNA-repelling surface are shown in blue.

Fig. 3.

Proposed plectonemic DNA compaction through H-NS. A, Model of H-NS superhelix (yellow: putative position of the wHTH domains; orange: H-NS_1–83 oligomer; blue: R15, R19, and K32) accommodating two DNA double helices (gray). For comparison the toroid compaction in the nuclear core particle is shown (green: histone core; red: DNA). B. 90° rotation about horizontal axis.

Fig. 4.

SAXS analysis of self-association at two temperatures. A, SAXS pattern calculated from an oligomer of four linked H-NS_1–83 dimers (green) and a single Site 1 H-NS_1–82 dimer (red), fitted to experimental data of 0.4 mM H-NS_1–83 recorded at 10 °C (χ = 5.8), and 40 °C (χ = 2.4). B, ∼90° views of 10 °C ab initio sphere models (gray) superimposed onto four linked H-NS_1–82 dimers (orange). C, ∼90° views of 40 °C ab initio sphere models (gray) superimposed onto one H-NS_1–82 site 1-linked dimer (cyan and blue). Ab initio models were produced based on corresponding SAXS data shown in A.