Poster abstracts
Poster number 14 submitted by Annie Ashraf
Towards Elucidating the Structure and Oligomeric State of the Membrane-Adjacent Domain of Cadherin-23, a Protein Essential for Hearing.
Qurat Ashraf (MCDB, The Ohio State University), Giovanna Grandinetti, Narui Yoshie, Elakkiya Tamilselvan, Travis Harrison-Rawn, Jasanvir Sandhu, Marissa Boyer, Marcos Sotomayor (The Ohio State University)
Abstract:
Mechanotransduction is the process by which hair cells of the inner ear transform mechanical stimuli into electrical signals. These electrical signals are transmitted to the central nervous system for processing in hearing and balance. Hair cells harbor actin-filled apical protrusions called stereocilia. Upon mechanical stimulation, fine “tip-link” protein filaments connecting the tip of a stereocilium to a neighboring taller stereocilium are stretched. As these filaments stretch, they pull and open ion channels to elicit an ionic current that initiates sensory perception. Tip-link filaments are made of homodimers of cadherin-23 (CDH23) and protocadherin-15 (PCDH15), two proteins involved in inherited deafness. CDH23 forms the upper two-thirds of the tip links and interacts with PCDH15 at the lower end to form a hetero-tetrameric complex. CDH23 and PCDH15, calcium-dependent cell-cell adhesion proteins, have multiple extracellular cadherin (EC) “repeats”, each about 100 amino acids long with a characteristic Greek-key fold. In addition to these EC repeats, both CDH23 and PCDH15 have membrane-adjacent domains, MAD28 and MAD12, respectively, which are predicted to be similar in structure. Intriguingly, biochemical assays and crystal structures of PCDH15 reveal that MAD12 induces parallel dimerization and that it has a ferredoxin-like fold, unlike the Greek key fold for EC repeats. Yet the structure and biochemical properties of MAD28 and of its adjacent CDH23 EC repeats are unknown. To determine whether CDH23 MAD28 has a ferredoxin-like fold that facilitates parallel dimerization, we tested several protocols to express and purify various CDH23 fragments that include EC25 to MAD28. We are now able to express and purify milligram amounts of well-refolded protein being used for biophysical characterizations using small-angle X-ray scattering and Cryogenic electron microscopy. To further our understanding of the role of MAD28 in mediating dimerization of CDH23, we introduced several mutations aimed to disrupt a possible MAD28 dimerization interface suggested by AlphaFold 2. Results from these experiments will advance our understanding of the role played by CDH23 in inner ear mechanotransduction.
References:
Jaiganesh, A. et al., (2018). Zooming in on cadherin-23: Structural diversity and potential mechanisms of inherited deafness. Structure, 25:1210-1225.
De-la-Torre, P. et al., (2018). A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15. Biophysics Journal, 115: 2368-2385.
Choudhary, D. et al., (2020). Structural Determinants of Protocadherin-15 Mechanics and Function in Hearing and Balance Perception. PNAS.
Keywords: Mechanotransduction, Tip link, Biophysical characterization