Abstract
Purpose :
We have identified steroidogenic acute regulatory domain protein 3 (StARD3) as the lutein-binding protein in the human retina. In the present work, we further crystalized this protein. In an effort to obtain molecular details of lutein in complex with StARD3, we also extended the resolution limit for a crystal structure of StARD3 and docked this with lutein.
Methods :
The binding domain of StARD3 protein (216-444 aa) was overexpressed in E. coli, and purified by ion-exchange and gel filtration chromatography. The purity and concentration of StARD3 were determined by SDS-PAGE and absorption spectroscopy, respectively. Crystals were grown by hanging drop vapor diffusion at room temperature by combining equal volumes of StARD3 protein and reservoir solution containing 0.1 M CHES buffer pH 8.9 to 9.6, 0.7-1.0 M K/Na tartrate, 0.2 M LiSO4, and 10 mM DTT. Crystals were frozen, and X-ray diffraction was measured at the SIBYLS beamline 12.3.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory. Initial phases were obtained by molecular replacement with the published structure (pdb id 1EM2, 2.2Å resolution) as a search model. The structure of StARD3 was refined using PHENIX, and lutein docking was performed by COOT.
Results :
SDS-PAGE revealed a single protein band of ~ 26 kD. The yield of protein was 2 mg pure protein for 1 L of culture. StARD3 crystals grew as hexagonal rods (20µm x 300µm) within 3-5 days. Crystals diffracted to the 1.74 Å resolution limit. Iterative refinement with phase extension yielded a model with R = 0.187 and Rfree = 0.203. One molecule of lutein could be docked into the hydrophobic tunnel of the StARD3 structure, guided by the locations of two molecules of CHES, which is consistent with the one-lutein-per-StARD3 protein stoichiometry determined by our previous SPR binding affinity study.
Conclusions :
We have extended phases for a crystal structure of the StARD3 lutein-binding domain to the 1.74 Å resolution limit, and lutein could be successfully docked into this crystal structure, offering a route to co-crystallization of StARD3 with lutein.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.