September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Crystal Structure of the Human Lutein-Binding Protein (StARD3) Binding Domain with Phase Extension to 1.74 Å Resolution
Author Affiliations & Notes
  • Binxing Li
    Department of Ophthalmology, Univ of UT Sch Med/Moran Eye Ctr, Salt Lake City, Utah, United States
  • Martin P. Horvath
    Department of Biology, University of Utah, Salt Lake City, Utah, United States
  • Evan W. George
    Department of Biology, University of Utah, Salt Lake City, Utah, United States
  • Quang Tran
    Department of Biology, University of Utah, Salt Lake City, Utah, United States
  • Saeed Shihab
    Department of Ophthalmology, Univ of UT Sch Med/Moran Eye Ctr, Salt Lake City, Utah, United States
  • Ty Mattinson
    Department of Ophthalmology, Univ of UT Sch Med/Moran Eye Ctr, Salt Lake City, Utah, United States
  • Paul S Bernstein
    Department of Ophthalmology, Univ of UT Sch Med/Moran Eye Ctr, Salt Lake City, Utah, United States
  • Footnotes
    Commercial Relationships   Binxing Li, None; Martin Horvath, None; Evan George, None; Quang Tran, None; Saeed Shihab, None; Ty Mattinson, None; Paul Bernstein, None
  • Footnotes
    Support  EY-11600, EY-14800, and an unrestricted departmental grant to the Moran Eye Center from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3624. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Binxing Li, Martin P. Horvath, Evan W. George, Quang Tran, Saeed Shihab, Ty Mattinson, Paul S Bernstein; Crystal Structure of the Human Lutein-Binding Protein (StARD3) Binding Domain with Phase Extension to 1.74 Å Resolution. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3624.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×