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.

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 24 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.

No abstract

A “lock and key” is the most common depiction of protein binding, protein has a specific structure, and its binding molecule of interest can fit into it. What happens when a protein domain needs to bind to multiple biomolecules? The transcription factor CRX is one of those with an activation domain that can bind to various biomolecules. CRX regulates the gene expression of cone and rod photoreceptors. To effectively regulate gene expression, CRX’s activation domain interacts with multiple transcriptional co‐activators. Unlike the “lock and key” concept with defined structures, we propose that the activation domain of CRX is intrinsically disordered, allowing for interactions of multiple proteins in the same domain. In support of a disordered activation domain, we observe that proline and serine amino acids are abundant in the CRX activation domain. We also note an abundance of pathogenic frameshift mutations and a lack of point mutants in the activation domain. To characterize the structure of CRX, we attempted to purify full‐length CRX and CRX coexpressed with known transcriptional coactivator NRL. We hypothesize that this latter experiment will enhance the stability and expression of full‐length CRX. We have purified CRX using nickel chromatography and are currently optimizing the next steps to enhance the purity of CRX and NRL. The information from biochemical and structural experiments on purified CRX will provide insight into the transcriptional regulation of CRX, leading to the development of photoreceptor neurons.