SAXS Examinations of the Redox-Dependent Formation of a DNA-SOD1 Complex.

Cu/Zn superoxide dismutase (SOD1) plays a key role in the maintenance of cellular reactive oxygen species (ROS) homeostasis as an antioxidant enzyme. We recently found that SOD1 is involved in the regulation of gene expression in response to changes in cellular ROS levels by binding to DNA-specific sequences. Moreover, the SOD1 binding to DNA was observed to be redox-dependent in solutions. Thus, we examined the redox-dependent DNA binding of SOD1 by multiple measurements, including small-angle X-ray scattering (SAXS), indicating the redox-dependent formation of a DNA-SOD1 complex in solutions. The redox-dependent formation of the DNA-SOD1 complex could underlie the SOD1 regulation of gene expression.

A new function of copper zinc superoxide dismutase: as a regulatory DNA-binding protein in gene expression in response to intracellular hydrogen peroxide.

In microorganisms, a number of metalloproteins including PerR are found to regulate gene expression in response to environmental reactive oxygen species (ROS) changes. However, discovery of similar regulatory mechanisms remains elusive within mammalian cells. As an antioxidant metalloenzyme that maintains intracellular ROS homeostasis, copper zinc superoxide dismutase (SOD1) has high affinity for DNA in solution and in cells. Here, we explored the regulatory roles of SOD1 in the expression of genes in response to ROS changes within mammalian cells. SOD1-occupied DNA sites with distinct sequence preference were identified. Changing ROS levels both were found to impact DNA-SOD1 interactions in solution and within HeLa cells. GGA was one of the base triplets that had direct contact with SOD1. DNA-SOD1 interactions were observed to regulate the ROS-responsive expression of functional genes including oncogenes and amyotrophic lateral sclerosis-linked genes in transcriptional phases. Our results confirm another function of SOD1, acting as a H2O2-responsive regulatory protein in the expression of numerous mammalian genes.

The Conformational Preference of Chemical Cross-linkers Determines the Cross-linking Probability of Reactive Protein Residues.

Chemical cross-linking mass spectrometry (XLMS) is an emerging technique in structural biology. Providing the cross-linked peptides are identified by mass spectrometry with high confidence, a distance restraint can be applied between the two reactive protein residues, with the upper bound corresponding to the maximal span of the cross-linker. However, as the upper bound is typically over 20 Å, cross-link distance restraints are unrestrictive and provide a marginal improvement in protein structural refinement. Here we analyze the experimental cross-links for lysine or acidic residues and show that the distribution of Cß-Cß' distances can be described with two overlapping Gaussian species. In addition to the pairwise occurrence probability of the reactive protein residues, we show that the distribution profile of the cross-link distances is determined by the intrinsic conformational propensity of the cross-linker. The cross-linker prefers either a compact or extended conformation and, once attached to a reactive protein residue, predominantly an extended conformation. Consequently, the long-distance Gaussian species occurs at a much higher probability than the short-distance species in the observed cross-links. Together, the probabilistic distribution of the cross-link distance allows the construction of a more restrictive restraint for structural modeling and better use of the XLMS data.