Cooperative Binding of PhoB^DBD to Its Cognate DNA Sequence—A Combined Application of Single-Molecule and Ensemble Methods

Abstract

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process.

Fluorescence‐Labeled Bis‐benzamidines as Fluorogenic DNA Minor‐Groove Binders: Photophysics and Binding Dynamics

“…[14][15][16][17] Single-molecule fluorescence techniques have emerged as powerful tools for characterization of the kinetics of biosupramolecular interactions [18] and have been successfully applied to study DNA binding with a number of ligands, mainly proteins. [19][20][21] Our FCS study with BBA-OG demonstrated that its association rate constants are in agreement with those reported for typical minor-groove binders, whereas the dissociation rates are much higher. [22][23][24][25] These results suggest that the selectivity of BBA-OG for A/T-rich sites mainly arises from the association process, whereas its low affinity constants result from its high dissociation rate constants, which are several orders of magnitude higher than those of typical minor-groove binders.…”

Interplay between genetic regulation of phosphate homeostasis and bacterial virulence

“…Pit does not require chemical energy to drive Pi transport when it is abundant, but when conditions are limiting, the cell uses ATP to scavenge Pi and similar P-containing compounds from the environment via the Pst system. 10 PhoB is a typical winged-helix response regulator that upon aspartyl phosphorylation forms a dimer, which binds in headto-tail arrangement to DNA sequences upstream of Pho regulon genes to recruit RNA polymerase (RNAP) [11][12][13][14] (Fig. 1).…”

The Influence of Repressor DNA Binding Site Architecture on Transcriptional Control

“…The recent structural characterization of full-length KdpE, another OmpR/PhoB family member, bound to its direct repeat site confirmed these different domain symmetries while identifying an additional level of asymmetry resulting from intramolecular contacts between the receiver and DNA binding domains within one KdpE subunit (18). Nevertheless, full-length ArcA-P has been reported to form oligomers (19), as have both the isolated N-terminal and C-terminal domains (16), suggesting that although the minimal DNA binding unit is likely a dimer, as demonstrated for PhoB and OmpR (20–22), oligomerization beyond a dimer may explain binding to multiple direct repeats.…”