DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

Abstract

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

Control of virulence gene transcription by indirect readout in Vibrio cholerae and Salmonella enterica serovar Typhimurium

“…Docking the Fis dimer successfully to its target involves an induced fit mechanism in which minor groove compression accompanied by bending of the target DNA accommodates the side-by-side protein monomers (Stella et al, 2010). Fis binding is influenced strongly by indirect readout and minor groove conformation (Hancock et al, 2016). Changing the supercoiling of the DNA also exerts an effect: Fis prefers DNA that is negatively supercoiled, a requirement that is easily met during the early stages of logarithmic growth when Fis is abundant.…”

Crystal structure of the nucleoid-associated protein Fis (PA4853) fromPseudomonas aeruginosa

“…Multiple structure-function studies have focused on Escherichia coli Fis (referred to as EcFis) and have shown that it has a four-helix bundle structure and functions as a homodimer (Kostrewa et al, 1991;Osuna et al, 1991;Stella et al, 2010;Shao et al, 2008;Hancock et al, 2013Hancock et al, , 2016. The disordered or flexible N-terminal region is required for Hinmediated inversion.…”

“…The HTH region is strongly conserved, suggesting that the DNA-binding properties of Fis are conserved. Several DNA-bound structures suggest that EcFis initially recognizes DNA targets with intrinsically narrow minor grooves using the separation between the HTH motifs in the EcFis dimer as a ruler (Stella et al, 2010;Hancock et al, 2013Hancock et al, , 2016.…”

Dissecting and analyzing key residues in protein‐DNA complexes

“…Further, simultaneous studies on folding and binding have been reported in Mutsα, a DBP . Recently, the role of DNA sequence on change in its structure and affinity for binding of DBPs has been reported . In addition, the disorderness is often observed in protein‐DNA complexes and about half of the proteins are predicted to be disordered among eukaryotic transcription factors.…”

“…32 Recently, the role of DNA sequence on change in its structure and affinity for binding of DBPs has been reported. 33 In addition, the disorderness is often observed in protein-DNA complexes and about half of the proteins are predicted to be disordered among eukaryotic transcription factors. Intrinsically disordered regions generally facilitate binding and folding not only in protein-DNA but also in protein-RNA and protein-protein interactions.…”