Heme inhibits the DNA binding properties of the cytoplasmic heme binding protein of Shigella dysenteriae (ShuS).
Heme uptake and utilization by pathogenic bacteria are critical for virulence and disease, since heme and heme proteins are a major source of iron within the host. Although the role of outer membrane heme receptors in this process has been extensively characterized at the genetic and biochemical level, the role of the cytoplasmic heme binding proteins is not yet clear. The Shigella dysenteriae cytoplasmic heme binding protein, ShuS, has previously been shown to promote utilization of heme as an iron source at low to moderate heme concentrations and to protect against heme toxicity at high heme concentrations. Herein, we provide evidence that ShuS of S. dysenteriae sequesters DNA non-sequence-specifically with a binding affinity of 3.6 microM as determined by fluorescence anisotropy studies. The ability to bind DNA was observed to be restricted to the apoprotein only. The molecular mass of the apo-ShuS-DNA complex was estimated to be approximately 700 kDa by size exclusion chromatography. Atomic force microscopy (AFM) revealed that apo-ShuS forms aggregates in the presence of DNA and provides a scaffolding matrix from which DNA is observed to loop outward. The AFM images of apo-ShuS-DNA complexes were strikingly similar to the AFM images of the stress-induced Escherichia coli protein, Dps, when complexed with DNA; however, unlike the Dps protein, ShuS failed to protect DNA against oxidative stress in vitro and in vivo. Since free heme can generate reactive oxygen species which are damaging to cellular DNA, the ability of ShuS to physically sequester DNA may provide a molecular basis for its role in preventing toxicity associated with high heme concentrations.
Kaur
Ajinder P
AP
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.
Wilks
Angela
A
D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D006418:Heme / Q000494:pharmacology*; D006420:Hemeproteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018384:Oxidative Stress / Q000187:drug effects; D012762:Shigella dysenteriae / Q000737:chemistry*
D004268:DNA-Binding Proteins; D006420:Hemeproteins; D006418:Heme; D004247:DNA