Knowledge Bank

Sound travels through the external and middle ear to the fluid-filled cochlea where mechanosensitive hair cells transform it into electrochemical signals. On the apical side of each hair cell, a set of hair-like protrusions, called stereocilia form a bundle with filamentous connections (tip links) that are essential for hearing. In response to the mechanical force generated by sounds waves, stereocilia move back and forth, thereby stretching tip links and opening nearby transduction channels. The tip link is formed by two non-classical cadherins, Cadherin-23 (CDH23) and protocadherin-15 (PCDH15), which are members of the cadherin superfamily of calcium-dependent adhesion proteins. CDH23 has 27 extracellular cadherin (EC) repeats and PCDH15 contains 11 EC repeats. Single missense mutations in PCDH15 are known to cause deafness, and absence of this protein leads to both deafness and blindness. A recent study showed that a point mutation (V767-) located in the PCDH15 EC7 repeat is pathogenic. My project focuses on determining the structure of the PCDH15 EC7 to EC8 fragment via X-ray crystallography, both to understand its function in hearing and to discover the structural effects of pathogenic mutations. In a first step to achieve these goals we used a DNA construct that encodes for the mouse PCDH15 EC7-8 fragment to express it in a bacterial system. After transformation and culture, we successfully observed protein expression. However, the protein fragment was not soluble and aggregated in inclusion bodies. Therefore, I did protein purification under denaturing conditions, followed by refolding and size-exclusion chromatography, and obtained pure folded protein amenable for crystallization. I obtained protein crystals that diffracted when exposed to X-rays and then refined the crystallization conditions to obtain a full data set with a resolution of 2.0 Å. Using this data set I found a molecular replacement solution for the first crystal structure of PCDH15 EC7-8, which I am currently refining. In this thesis I will describe each of the steps involved in obtaining the structure of PCDH15 EC7-8 and will discuss how the study of this structure will help us understand the mechanisms of mechanotransduction in normal and impaired hearing.