Structural basis of functional alterations caused by mutations in the homeodomain of CRX

Abstract

Purpose : CRX is a retina-specific transcription factor that controls photoreceptor differentiation and gene expression. When mutated CRX causes human retinopathies. Certain mutations occur in conserved amino acids (R41 and R90) within the homeodomain (HD). Biochemical evidence has shown that they can alter CRX-HD binding to DNA and modify its activity in vitro. Here, we address the impact of the mutations on the structure-function of CRX-HD.

Methods : Hexa-histidine labeled CRX-HD and CRX-HD^R41Q were expressed in E. coli and purified using affinity and size exclusion chromatography (SEC). The secondary structures and thermal stabilities were evaluated +/- DNA (BAT1 from the rhodopsin promoter) using circular dichroism spectroscopy (CD). The oligomeric states of proteins were determined by SEC and by analytical ultracentrifugation (AUC). For some experiments, proteins were uniformly labeled with ¹⁵N/¹³C. NMR spectra (1D/2D ¹H-NOESY, ¹³C- and ¹⁵N-HSQC, HN(CA)CO, HN(CO)CA, HCCH-COSY) were collected on a Bruker 800 MHz spectrometer. Protein binding to BAT1 was measured by biolayer interferometry (BLI) and NMR.

Results : The CRX-HD and CRX-HD^R41Q proteins exhibited very similar CD spectra and thermal stabilities when free in solution, indicating that the overall folding of the HD was not significantly altered. However, CRX-HD^R41Q was significantly less stable when bound to DNA compared to CRX-HD. Both HDs were primarily monomeric in solution, as judged by SEC and AUC. Both HDs bound to BAT1 stoichiometrically. However, the R41Q substitution reduced the binding affinity to BAT1. Moreover, the interaction of the CRX-HD^R41Q with BAT1 exhibited significant changes in the NMR spectra in the core TAAT sequence. Analysis of the protein-DNA complexes at steady state suggests that, while two molecules of CRX-HD bind to BAT1, only one molecule of CRX-HD^R41Q binds per BAT1.

Conclusions : These results demonstrate that the R41Q mutation disturbs the interaction of the CRX homedomain by reducing the occupancy of the DNA cis-element BAT1. This change in protein-DNA interactions could cause a change in transcriptional activation or specificity of cis-element selection, potentially contributing to disturbance of retinal gene expression.

This is a 2020 ARVO Annual Meeting abstract.