Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein

Abstract

In Bacillus subtilis, PerR is a metal-dependent sensor of hydrogen peroxide. PerR is a dimeric zinc protein with a regulatory site that coordinates either Fe(2+) (PerR-Zn-Fe) or Mn(2+) (PerR-Zn-Mn). Though most of the peroxide sensors use cysteines to detect H(2)O(2), it has been shown that reaction of PerR-Zn-Fe with H(2)O(2) leads to the oxidation of one histidine residue. Oxidation of PerR leads to the incorporation of one oxygen atom into His37 or His91. This study presents the crystal structure of the oxidized PerR protein (PerR-Zn-ox), which clearly shows a 2-oxo-histidine residue in position 37. Formation of 2-oxo-histidine is demonstrated and quantified by HPLC-MS/MS. EPR experiments indicate that PerR-Zn-H37ox retains a significant affinity for the regulatory metal, whereas PerR-Zn-H91ox shows a considerably reduced affinity for the metal ion. In spite of these major differences in terms of metal binding affinity, oxidation of His37 and/or His91 in PerR prevents DNA binding.

Mutational Analysis of the Metal-binding Sites of Peroxide Sensor PerR

“…13 Furthermore, these results are consistent with the previous observation that H37A mutant and PerR with oxidized H37 retain Mn 2+ -binding ability but not H91A mutant and PerR with oxidized H91. 16 Thus our results strongly support the previous hypothesis that the oxidation of H91 leads to an inactive PerR through loss of metal binding at the regulatory metal binding site. Our results also support the overall idea that the inactivation of PerR by H37 oxidation proceeds through loss of proper coordination by H37.…”

“…It is likely that the relatively low level of H91 oxidation in H37A mutant is caused by the observed decrease in metal binding affinity of H37A mutant described previously. 16 The total absence of histidine oxidation in the H91, H93 and D104 strongly suggest that mutations at the C-terminal metal binding site preclude these mutants binding Fe 2+ .…”

“…Thus, it is likely that these mutant PerR proteins also adopt a conformation similar to apo-PerR and oxidized PerR which are poorly suited for DNA binding. [15][16] In this conformation the regulatory metal binding site is separated into two parts, the N-terminal domain containing H37 and D85 and C-terminal domain containing H91, H93 and D104 (Fig. 1B).…”

The Fur homologue BosR requires Arg39 to activate rpoS transcription in Borrelia burgdorferi and thereby direct spirochaete infection in mice

“…Pfam alignments of FUR proteins (PF01475) show that BosR's Arg39 aligns with a highly conserved arginine present in virtually all FUR proteins. X-ray crystallography studies show that FUR proteins consist of two domains: an N-terminal DNA-binding domain and a C-terminal dimerization domain Butcher et al, 2012;Dian et al, 2011;Gilston et al, 2014;Jacquamet et al, 2009;Lin et al, 2014;Lucarelli et al, 2007;Makthal et al, 2013;Pohl et al, 2003;Sheikh & Taylor, 2009;Shin et al, 2011;Traoré et al, 2006Traoré et al, , 2009. The DNA-binding domain contains a winged helix-turn-helix (wHTH) motif that provides the protein with the means to bind DNA (Pohl et al, 2003).…”

Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips

“…It is unclear if cells simply tolerate such damages on histidines or employ active mechanisms to recognize them and use them as redox sensors or as damage markers for promoting protein degradation. The only known biological function of 2-oxohistidine is to serve as a redox sensor on bacterial transcription factor PerR (18), whereas other studies have used 2-oxohistidine as a stable marker of protein damage during oxidative stress (12,19).…”