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Ravi Prakash Yadav, Liping Yu, Nikolai Artemyev; Delineation of the Farnesyl-Binding Site on AIPL1 by NMR. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1743.
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© ARVO (1962-2015); The Authors (2016-present)
AIPL1 is a specialized chaperone of phosphodiesterase-6 (PDE6) in rods and cones. Mutations in AIPL1 lead to destabilization of PDE6 and cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Binding of the prenylated C-termini of PDE6 to the AIPL1 FKBP domain is critical to AIPL1 chaperone function. We have investigated the farnesyl-binding site of AIPL1 by NMR.
The uniformly 15N- and selectively 13C-methyl-labeled AIPL1 FKBP domain was obtained by growing E. coli with 15NH4Cl, 13C-α-ketobutyrate, and 13C-α-ketoisovalerate. The 15N/1H and 13C/1H HSQC spectra were collected for the purified AIPL1 FKBP and select mutants in the absence and presence of S-farnesyl-L-cysteine methyl ester (FC). The FC-binding site was determined and the FC ligand was docked to the homology model of the AIPL1 FKBP using the restraint data obtained from the NMR experiments. Binding of FC to the AIPL1 FKBP and its mutants was also assessed using FRET.
Binding of FC caused selective changes in the 15N/1H HSQC spectra as well as in the 13C/1H HSQC spectra of the Ile CδH3 and Leu/Val methyl regions of AIPL1 FKBP. The Ile and Leu methyl peaks that shifted upon FC binding were assigned based on the NMR analysis of select Ile→Leu and Leu→Ile mutants of the AIPL1 FKBP.
The NMR analysis delineated a novel prenyl-binding site at the interface of the unique insert region and the core FKBP fold of AIPL1. A model of the complex of AIPL1 FKBP with FC generated from this study facilitates identification of LCA4 mutations perturbing the ligand-binding to AIPL1.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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