EFFECT OF DISTAL MUTATIONS IN FORCE-FILTERING OF MECHANOSENSING PROTEIN COMPLEX

Abstract

In physiology, many mechanosensing proteins act as a force filter. They transduce or dissipate mechanical cues depending on the amplitude or intensity of the signal. Numerous mutations found in these proteins can alter their interaction networks and change their force response, often leading to disease phenotypes. For example, mutations in titin are associated with cardiomyopathies, and mutations in tip-link proteins are linked to HL. My work involves studying the tip link, which serves as a force filter for sound stimuli. There are mutations in tip-link protein Cdh23 that are directly associated with progressive HL (PHL). Most studies have focused on mutations in the binding domains of Cdh23 due to their critical role in maintaining the integrity of the tip-link complex. However, the role of distal mutations is less explored, despite their higher prevalence. So, my overall objective was to understand how the distal mutations affect the force-filtering ability of the tip-link. Based on prevalence and severity, I identified two mutations, P217L and R278Q, located in the 3rd extracellular domain of Cdh23. Interestingly, the homozygous [P217L]:[P217L] mutation leads to congenital HL, whereas the heterozygous [P217L]:[WT] and compound heterozygous [P217L]:[R278Q] mutations are associated with progressive hearing loss (PHL). These mutations do not affect the formation of the binding interface as the people with these mutations can hear sounds as long as the regeneration of hair cells but can alter the force response of Cdh23. I hypothesize that these distal mutations although do not affect the formation of tip-link significantly, can alter the elasticity component which is essential for force-dissipation and hair-cell integrity. I used the magnetic tweezer and AFM-based force clamp experiment to measure the folding dynamics and bond lifetime dynamics of the WT and mutant complex. I observed that mutants have faster unfolding and slower refolding kinetics than WT, indicating that mutants are more sensitive to force than WT protein. AFM experiment shows that P217L mutation affects the binding affinity of Cdh23 with Pcdh15 by abolishing the slip-to-catch transition observed in WT and R278Q mutant tip-link complex. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulation show that mutants have altered interaction networks (H-bond and salt-bridge) in the non-interacting domains of Cdh23 as well as at the binding interface. Furthermore, the network analysis reveals that mutants have a narrower distribution of suboptimal paths in Cdh23 domains while the WT has a wider distribution of suboptimal paths. XIII This narrow distribution of paths in mutants makes the mutants easily malleable to force by reducing the force-bearing or force-dissipation ability of the Cdh23. Overall, my finding indicates that mutations located in non-interacting domains of Cdh23 affect the force-filtering of tip-link.