Dimerization and Heme Binding Are Conserved in Amphibian and Starfish Homologues of the microRNA Processing Protein DGCR8

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

Towards a Pharmacophore for Amyloid

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases

Extinction coefficients for homologous HBDs.

<p>Extinction coefficients for homologous HBDs.</p

The bat star DGCR8 HBD binds heme as a dimer.

<p>(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. <i>Inset</i>, a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) image of the 13.8-mL peak fraction of bat star HBD.</p

MALDI-TOF mass spectrometry analysis of crystals obtained from frog DGCR8 HBD.

<p>Ions with a “+1” charge state are labeled with their corresponding <i>m/z</i> values. The ion with <i>m/z</i> of 6820.0 roughly corresponds to the dimerization domain observed in the crystal structure.</p

Domain structure of DGCR8 and sequence alignment of the heme-binding domains.

<p>(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.</p

Crystal structure of the frog DGCR8 dimerization domain.

<p>(A–B) 2F_o-F_c 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.</p

Crystallographic statistics of the structure of the frog DGCR8 dimerization domain.

<p><i>R</i>_sym = ∑<i>_hkl</i>∑<i>_i</i> |<i>I_i</i>(<i>hkl</i>)−<<i>I</i>(<i>hkl</i>)> |/∑<i>_hkl</i> ∑<i>I_i</i>(<i>hkl</i>). <i>R</i>_work = ∑|<i>F_o</i>–<i>F_c</i>|/∑<i>F_o</i>. <i>R</i>_free = ∑|<i>F_o</i>−<i>F_c</i>|/∑<i>F_o</i>, calculated using a random set containing 10% reflections that were not included in refinement.</p

Part 1. High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for Accurate Biomolecule Characterization

Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values