Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity

Abstract

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF–DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: ‘associated’ (73° of DNA bend), ‘half-wrapped’ (107°) and ‘fully-wrapped’ (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

Structural interplay between DNA-shape protein recognition and supercoiling: the case of IHF

“…Six different topoisomers (ΔLk=-2,-1,0,1,2,3) of DNA minircircles containing 336 bp were constructed in order to achieve a similar σ range to the one observed in vivo (from -0.067 to +0.094). Then, these were attached to IHF via only its protruding β -ribbon arms to simulate how DNA spontaneously wraps around the protein following an initial bound state, which resembles an encounter complex formed at the beginning of the recognition process (Figure 1) [56, 14, 10].…”

“…The constructs were solvated using implicit generalized Born model at a sodium chloride salt concentration of 0.2 M with GBneck2 corrections, mbondi3 Born radii set and no cut-off for a better reproduction of molecular surfaces, salt bridges and solvation forces [44, 45, 46]. Langevin dynamics was employed for temperature regulation at 300 K with a collision frequency of 0.01 ps 1 in order to reduce the effective solvent viscosity and, thus, accelerate the exploration of conformational space [47, 10]. The protein and DNA were represented by ff14SB [48] and BSC1 [49] force fields, respectively.…”

“…The protein and DNA were represented by ff14SB [48] and BSC1 [49] force fields, respectively. Prolines were kept intercalated by restraining the distances between key atoms in the proline side chain and neighboring bases [10]. Following our protocols for minimization and equilibration [10], three replica simulations of 30 ns each were performed for each topoisomer with IHF bound, and three more for the same systems with the protein removed.…”

“…As has been described previously [10], interactions between IHF and the lateral DNA arms can be divided into four regions based on their position relative to the center of the binding site and the protein subunit to which the involved amino acid belongs. On the left-hand side (containing the A-tract), the α subunit is closer to the center and thus constitutes the 'near left' site, while the β subunit is farther and composes the 'far left'.…”

“…We furthermore observed the formation of large DNA–IHF aggregates in AFM images and the bridging of two DNA duplexes by a single IHF protein in MD simulations (See Figure 1) [10]. This condensating behavior is of particular importance to bacterial biofilms because IHF is located at crossing points in the extracellular DNA lattice [20] and is crucial to biofilm stability [21].…”