Abstract
Purpose :
AIPL1 is a specialized chaperone of phosphodiesterase-6 (PDE6) in rods and cones. Mutations in AIPL1 cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. The AIPL1-FKBP domain contributes to the chaperone activity by binding the C-terminal prenyl modifications of PDE6. Here, we have investigated the role of the AIPL1-TPR domain
Methods :
X-ray crystallography was used to solve the crystal structure of the AIPL1-TPR domain. NMR spectroscopy was employed to make the backbone and sidechain assignments of the TPR domain of AIPL1 by using an uniformly 15N- and 13C-labeled AIPL1-TPR obtained from E. coli cell culture grown in a minimal medium containing 15NH4Cl and 13C6-glucose. Then, NMR chemical shift mapping technique was used to determine the PDE6 regulatory Pγ-subunit binding site on AIPL1-TPR. Fluorescence-binding assays and biolayer interferometry were used to localize the AIPL1-TPR binding site on the Pγ-subunit. Dynamic light scattering was also used to study thermal stability of AIPL1 in the presence of Pγ peptides
Results :
We identified a novel interaction between the AIPL1-TPR domain and the regulatory Pγ-subunit of PDE6. The AIPL1-TPR binding site of Pγ was localized to the C-terminal region of Pγ with the residues Pγ70-74 playing a critical role in the interaction. A working model of the complex of AIPL1-TPR with the Pγ C-terminus has been generated by mapping the chemical shift perturbations of AIPL1-TPR observed upon titrating Pγ onto the atomic structure of the TPR domain. Currently, this model is being probed with mutational analysis of the partner proteins
Conclusions :
The interaction between the AIPL1-TPR domain and the C-terminal region of Pγ is likely to be critical for the chaperone function of AIPL1, since Pγ serves as a potent co-chaperone with AIPL1 in the folding and assembly of PDE6
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.