Senturia R et al. (2012), Dimerization and heme binding are conserved in ...

XB-ART-45592

PLoS One 2012 Jan 01;77:e39688. doi: 10.1371/journal.pone.0039688.

Show Gene links Show Anatomy links

Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.

Senturia R , Laganowsky A , Barr I , Scheidemantle BD , Guo F .

???displayArticle.abstract???
Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

???displayArticle.pubmedLink??? 22768307
???displayArticle.pmcLink??? PMC3388087
???displayArticle.link??? PLoS One
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: dgcr8 hbd

???attribute.lit??? ???displayArticles.show???

	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.

References [+] :

Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed