Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies

Alok K Sharma; Tobias Krieger; Alan C Rigby; Israel Zelikovic; Seth L Alper

doi:10.1016/j.bbrep.2016.08.022

Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies

Biochem Biophys Rep. 2016 Aug 26:8:184-191. doi: 10.1016/j.bbrep.2016.08.022. eCollection 2016 Dec.

Authors

Alok K Sharma¹, Tobias Krieger^{1

2}, Alan C Rigby³, Israel Zelikovic⁴, Seth L Alper¹

Affiliations

¹ Division of Nephrology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02215, United States.
² Paracelsus Medizinische Universitat, Salzburg, Austria.
³ Warp Drive Bio., Cambridge, MA 02139, United States.
⁴ Laboratory of Developmental Nephrology, Rambam Medical Center, and Department of Physiology and Biophysics, Faculty of Medicine, Technion, Haifa, Israel.

Abstract

Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) cause Pendred Syndrome /DFNB4, syndromic deafness with enlargement of the vestibular aqueduct and low-penetrance goiter. Here we present data on cloning, protein overexpression and purification, refolding, and biophysical characterization of the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS). We report a reproducible protein refolding protocol enabling milligram scale expression and purification of uniformly ¹⁵N- and ¹³C^/15N-enriched hPDS STAS-ΔIVS domain suitable for structural characterization by solution NMR. Circular dichroism, one-dimensional ¹H, two-dimensional ¹H-¹⁵N HSQC, and ¹H-¹³C HSQC NMR spectra confirmed the well-folded state of purified hPDS STAS-ΔIVS in solution. Heteronuclear NMR chemical shift perturbation of select STAS-ΔIVS residues by GDP was observed at fast-to-intermediate NMR time scales. Intrinsic tryptophan fluorescence quench experiments demonstrated GDP binding to hPDS STAS-ΔIVS with K_d of 178 μM. These results are useful for structure/function characterization of hPDS STAS, the cytoplasmic subdomain of the congenital deafness protein, pendrin, as well as for studies of other mammalian STAS domains.

Keywords: CD, Circular dichroism; Fluorescence; HSQC, Heteronuclear single quantum correlation spectroscopy; IVS; IVS, Intrinsic variable sequence; MS, Mass spectroscopy; NMR; Protein refolding; SLC26A4; STAS domain; hPDS, Human pendrin polypeptide.