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.2022 Aug;90(8):1584-1593.
doi: 10.1002/prot.26332. Epub 2022 Mar 23.

Structural and functional analysis of the human cone-rod homeobox transcription factor

Affiliations

Structural and functional analysis of the human cone-rod homeobox transcription factor

Penelope-Marie B Clanor et al. Proteins.2022 Aug.

Abstract

The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle X-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina-specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.

Keywords: CRX; homeodomain; photoreceptor; small-angle X-ray scattering.

© 2022 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

FIGURE 1
FIGURE 1
Human CRX protein organization and sequence analysis. (A) Cartoon showing the domain structure of human CRX (referred to as full‐length CRX) and the DNA‐binding domain of CRX (CRX‐DBD). Numbers indicate the amino acids positions encompassed in each construct. (B) Disorder prediction of the full‐length CRX sequence. The data from IUPred2A shown in red, from ODIN shown in blue, and from PrDOS in gray (see methods for details). (C) Distribution of clinically relevant mutations in the human CRX protein. Stop, missense, and frameshift mutations in human CRX protein were obtained from the ClinVar database. The distribution of mutations was plotted by amino acid position in the human CRX protein with cyan indicating the DNA‐binding domain and salmon box indicating the activation domain. The DBD contains 13 pathogenic missense mutations and three pathogenic frameshift mutations. The activation domain contains three pathogenic missense mutations and 25 pathogenic frameshift mutations. (D) Amino acid bias within the domains of CRX. The cyan box indicates the DNA‐binding domain and the salmon box indicates the activation domain
FIGURE 2
FIGURE 2
Solution properties of the CRX‐DBD peptide (A) Elution of CRX‐DBD from the size exclusion column. The peptide eluted as a single peak with a calculated molecular weight of 6.3 kDa. These data are consistent with a CRX‐DBD monomer. (B) Melting curves of CRX‐DBD at 222 nm. Data were globally fit in Calfitter to an equilibrium model and the lines show the fitted data. The calculated apparent melting temperature was 56.1 ± 1 °C. The SSR for the fit was 0.00938. (C) Predicted CD spectra for RCSB ID 2DMS, 3A01, 1FJL, 3CMY, 2MGQ (gray), and 1TF6 and 3EXJ (red lines) overlaid on a representative CD spectrum collected on the CRX‐DBD peptide (black points). (D) Predicted CD spectrum for the CRX‐DBD homology model (gray line) overlaid on the average CD spectrum for CRX‐DBD peptide (black points). Error bars are the SD of the measurements
FIGURE 3
FIGURE 3
SAXS analysis of CRX‐DBD (A) UV chromatogram from SEC‐SAXS of CRX‐DBD peptide. Inset shows the peak and the calculated molecular weight from multi‐angle light scattering (points) (B) log of intensity versus momentum transfer for the averaged frames containing CRX‐DBD (C) Pair‐distance distribution plot (D) Kratky plot of CRX‐DBD
FIGURE 4
FIGURE 4
Fitting of CRX‐DBD SAXS data to model structure (A) Fitting of the FOXS predicted SAXS intensity curve for RCSB ID 1FJL (χ2 = 0.99), 3A01 (χ2 = 0.99), and 3CMY (χ2 = 1.02) to the CRX‐DBD intensity data. (B) Envelope of the dummy atom model produced by DAMMIF aligned to the CRX‐DBD homology model (C) Electron density map produced from DENSS aligned to the CRX‐DBD homology model
FIGURE 5
FIGURE 5
CRX binds evolutionarily conserved CREs in the human genome. UCSC genome browser visualizations of the human (A)RHO and (B)PDE6B loci aligned with BAM density plots from an adult human retina CRX ChIP‐seq experiment identifying CRX‐binding regions (CRBs) in both loci10. Sequences highly conserved between vertebrate species are indicated as green peaks in the PhastCons Conservation data track. (C) Sequences of dsDNA probes used for EMSAs in theRHO andPDE6B promoter regions. Five base pair predicted CRX motifs within CBRs are indicated in blue lettering. Mutated bases are shown in red and capital letters to indicate the position and identity of the change. EMSA assays for (D) theRHO promoter and (E) thePDE6B promoter. 34 ng of was used for each assay, and 300 ng purified human MBP‐CRX‐DBD protein added to lanes indicated with +
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