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Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural
Alok K Sharma1, Tobias Krieger1,2, Alan C Rigby3
1Division of Nephrology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02215, United States.
Biochemistry and Biophysics Reports
|September 29, 2017
Summary
Mutations in the human Pendrin STAS domain (hPDS STAS-ΔIVS) are linked to Pendred Syndrome. This study details its protein refolding and characterization, revealing GDP binding crucial for understanding congenital deafness.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) are associated with Pendred Syndrome and DFNB4, characterized by hearing loss and vestibular aqueduct enlargement.
- The STAS (Sulfate Transporter Anion Transporter Sigma) domain is a key cytoplasmic subdomain of pendrin, implicated in its function and associated genetic disorders.
Purpose of the Study:
- To clone, overexpress, purify, and biophysically characterize the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS).
- To establish a reproducible protein refolding protocol for milligram-scale expression and purification of uniformly enriched hPDS STAS-ΔIVS for structural studies.
- To investigate the interaction of hPDS STAS-ΔIVS with GDP using biophysical and NMR techniques.
Main Methods:
- Recombinant protein expression, purification, and refolding of the hPDS STAS-ΔIVS domain.
- Circular dichroism (CD) spectroscopy for secondary structure assessment.
- Nuclear Magnetic Resonance (NMR) spectroscopy (1D 1H, 2D 1H-15N HSQC, 2D 1H-13C HSQC) for structural integrity and interaction studies.
- Heteronuclear NMR chemical shift perturbation (CSP) experiments to map GDP binding sites.
- Intrinsic tryptophan fluorescence quench experiments to determine binding affinity (Kd).
Main Results:
- A reproducible protocol was developed for milligram-scale expression and purification of refolded hPDS STAS-ΔIVS.
- CD and NMR spectra confirmed the well-folded state of the purified protein in solution.
- NMR CSP experiments indicated GDP binding to specific residues of hPDS STAS-ΔIVS.
- Fluorescence spectroscopy revealed GDP binding with a Kd of 178 μM.
Conclusions:
- The study successfully produced and characterized the recombinant hPDS STAS-ΔIVS domain, suitable for structural analysis.
- GDP binding to the hPDS STAS-ΔIVS domain was demonstrated, providing insights into its functional mechanism.
- These findings contribute to the structure-function understanding of pendrin and other mammalian STAS domains, relevant for congenital deafness research.
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