Understanding the Structure and Function of Proteins Essential for Hearing
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Date
2019-12
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The Ohio State University
Abstract
The ability to communicate is one the most fundamental skills attributed to the success of humans. Underlying this skill is the ability to hear, a process involving the conversion of mechanical stimuli, sound waves, into electrical impulses that are interpreted by the brain. This process is called mechanotransduction. An in-depth understanding of the different mechanisms and proteins involved in hearing will allow further action in aiding those living with hearing impairment or deafness.
Mechanotransduction takes place in the organ of Corti, housed along the length of the cochlea in the inner ear. This region is home to the inner (IHC) and outer hair cells (OHC), atop which bundles made of stereocilia are arranged in a staircase-like fashion. Connecting the top of the shorter stereocilium to the side of its taller neighbor is a protein filament called the tip link. The tip link consists of two proteins, protocadherin-15 and cadherin-23, interacting tip-to-tip to form a heterotetrameric complex that is essential for hearing. As mechanical stimuli act on hair cells, stereocilia move, pulling on the tip link, which opens an ion channel via interactions with protocadherin-15 and the tetraspan membrane protein of hair cell stereocilia (TMHS). Interestingly, while the channel opening in IHC leads to sensory perception, the same process in OHC results in voltage change that activates prestin, a motor protein to help amplify sound. Prestin is essential for this process, changing outer hair cell size based on changes in voltage. All the proteins and processes mentioned above are essential for hearing and much has been learned about the genes and proteins involved. However, little is known about the molecular mechanisms underlying mechanotransduction and sound amplification in the inner ear. Here I present our work on determining the structure of fragments of cadherin-23, on simulations of the complex formed by protocadherin-15 and TMHS, and on simulating a homology model of prestin.
Protocadherin-15 and cadherin-23 are made up of 11 and 27 extracellular cadherin (EC) repeats in addition to a 12th and 28th membrane adjacent domain (MAD), respectively. The structures for each protocadherin-15 EC repeat have been solved; however, there are multiple cadherin-23 fragments for which structures are unknown including EC10-11 and the linker region between EC11-12. Using dilution-refolding techniques and size-exclusion chromatography, I refolded, purified, and crystallized cadherin-23 EC11-12. Similar work has been performed for cadherin-23 EC10-11. Solving the structures of these EC repeats will help create a complete model of the tip link and will provide insights into the structural basis of inherited deafness.
Using molecular dynamics (MD) simulations, further mechanisms of the hearing process can be explored. Steered MD simulations are used to determine the strength of interactions within and between proteins. The tip link is thought to be responsible for the opening of the hair-cell ion channel through the interaction between protocadherin-15 and TMHS following force sensation via an unknown mechanism. As part of a group effort to understand how protocadherin-15 and TMHS respond to force, myself and many other members of the Sotomayor laboratory have used molecular dynamics simulations to stretch different models of the protocadherin-15-TMHS complex. My work focuses on molecular dynamics simulations of dimeric protocadherin-15 EC9-11 and MAD12 in complex with TMHS without calcium. These simulations will provide information on the strength of internal interactions in protocadherin-15, its interaction with TMHS, and on the mechanisms TMHS uses to open the TMC1 and TMC2 ion channels.
Last, I used a homology model of prestin and a coarse-grained MD simulation with a polarizable force field to perform long timescale simulations under the application of voltage. These simulations were carried out to learn the mechanism by which prestin mediates shape changes of the OHCs. The simulations show anions interacting with the protein under the application of 500 and 250 mV during multi-microsecond simulations. Further analysis is needed to determine changes of prestin’s volume over time to possibly determine a mechanism for OHC electromotility. Overall, the work presented here provides insight into three important roles of inner-ear mechanotransduction: force propagation through cadherin-23, channel gating, and prestin-mediated sound amplification.
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Keywords
Hearing, Crystallography, Mechanotransducion, Cadherin-23, Prestin, Molecular Dynamics Simulations, Protocadherin-15, Tetraspan membrane protein of hair cell stereocillia (TMHS)