XB-ART-45592PLoS One. January 1, 2012; 7 (7): e39688.
Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.
PubMed ID: 22768307
PMC ID: PMC3388087
Article link: PLoS One.
Grant support: GM080563 NIGMS NIH HHS , R01 GM080563 NIGMS NIH HHS
Genes referenced: dgcr8 hbd
Article Images: [+] show captions
|Figure 1. Domain structure of DGCR8 and sequence alignment of the heme-binding domains.(A) Domain structure of human DGCR8 and schematics of the NC1 and HBD constructs used in this study. (B) Schematic of how the DGCR8 HBD binds Fe(III) heme. (C) Sequence alignment of bat star, frog and human HBDs. Identical residues are shaded in black. Residues that are identical only between two species are shaded in gray. Red stars denote residues in human HBD known to be important for heme binding. Secondary structure assignments derived from the crystal structure of frog dimerization domain are shown below the sequences, with β-strands as green arrows and loops as bars.|
|Figure 2. The bat star DGCR8 HBD binds heme as a dimer.(A) Electronic absorption spectrum of bat star HBD. Peak wavelengths and the corresponding extinction coefficients are labeled. (B) Size exclusion chromatogram of the bat star HBD, obtained from the last step of the purification procedure. The elution volumes of the dimeric human HBD (54 kDa) and the monomeric human NC9 (29 kDa) proteins are indicated as triangles. Inset, a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) image of the 13.8-mL peak fraction of bat star HBD.|
|Figure 3. Crystal structure of the frog DGCR8 dimerization domain.(A–B) 2Fo-Fc electron density maps, contoured at 1σ level, of the N- and C-terminal regions of the frog dimerization domain, respectively. (C) Wall-eyed stereo diagram of the crystal structure of frog dimerization domain. The dimer subunits are colored green and blue. Secondary structures from the green subunit are denoted with a prime. The crystallographic two-fold axis relating the two subunits is indicated by the arrow. Residues known to be important for heme binding are highlighted in red. (D) Superimposition of human (orange) and frog (blue) dimerization domain Cα traces shown in stereo.|
|Figure 4. MALDI-TOF mass spectrometry analysis of crystals obtained from frog DGCR8 HBD.Ions with a “+1” charge state are labeled with their corresponding m/z values. The ion with m/z of 6820.0 roughly corresponds to the dimerization domain observed in the crystal structure.|