DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-ligated Heme Protein

Ian Barr,Aaron T Smith,Rachel Senturia,Yanqiu Chen,Brooke D Scheidemantle,Judith N Burstyn,Feng Guo

doi:10.1074/jbc.m110.180844

Abstract

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.

Highlights

Heme proteins use a variety of amino acids to serve as the axial ligands for the heme iron
We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli
In cytochrome P450, ferrous heme ligated by a cysteine thiolate trans to CO exhibits a spectrum with the 366-nm band substantially less intense than the ϳ450-nm peak

Summary

EXPERIMENTAL PROCEDURES

Reagents—Horse skeletal muscle apomyoglobin was from Sigma-Aldrich (St. Louis, MO). The purified protein was exchanged into desired buffers for spectroscopic studies, as indicated in the figure legends, using centrifugal concentrators with a molecular mass cut-off of 30 kDa. SeMet-labeled frog DGCR8 HBD-His was expressed using the same vector, host strain, and procedure as described by Van Duyne et al [23]. Prior to the mercury titration experiments, the purified HBD mutant was concentrated and exchanged into a buffer containing 50 mM EPPS (pH 8.0) and 400 mM NaCl, using a centrifugal concentrator with a molecular mass cut-off of kDa. The extinction coefficient of the heme-bound hHBDC430S protein (⑀280 nm ϭ mMϪ1 cmϪ1) was determined experimentally by measuring its absorption at 280 nm and its protein concentration using the Micro BCA protein assay kit (Pierce). The reactions were analyzed using denaturing 15% polyacrylamide gels and autoradiography

RESULTS

D M Expressed without δ-ALA

DISCUSSION