Inner-ear Mechanotransduction Research Articles

In silico study of CIB N-myristoylation's role in inner-ear mechanotransduction.

Inner-ear mechanotransduction is initiated when mechanical stimuli from sound and head movements stretch tip-link protein filaments to open ion channels that trigger sensory perception. These inner-ear mechanotransduction channels are formed by transmembrane channel-like (TMC) proteins 1 and 2 in complex with calcium and integrin-binding (CIB) proteins 2 and 3. Interestingly, CIB proteins may be myristoylated at their N-terminal glycine following removal of their first methionine. This addition of a 14-carbon unsaturated fatty acid is known as N-myristoylation and may provide an anchor point that mediates association with biological membranes. However, whether this is relevant for CIB function in TMC-mediated mechanotransduction is unclear. Here, we use experimentally supported AlphaFold 2 models of full-length TMC1 and TMC2 in complex with CIB2 and CIB3 in all-atom molecular dynamics simulations aimed at understanding the functional role of CIB myristoylation in TMC activation. These simulations show that the myristoylated N-terminus of CIB facilitates association of the complex with the lipid bilayer. Similarly, an amphipathic TMC α0 helix inserts into the membrane. Close association of the complex with the lipid bilayer suggest that membrane tension might play a role in the activation of TMC/CIB complexes in the inner ear.

Calcium-dependent strength of the inner-ear cadherin-23 ectodomain.

Cadherin proteins are essential for multicellular organisms and are involved in multiple processes including calcium-dependent cell-cell adhesion and inner-ear mechanotransduction. Cadherin-23 is an integral component of a fibrous protein filament, called the tip link, that is ultimately responsible for converting the mechanical energy of sound into the opening and closing of a gated ion channel that triggers signaling through the auditory nerve. Proper functioning of the tip link depends on its mechanical strength, which is stabilized by interactions of calcium ions with nearby protein residues at discrete triplet binding sites between extracellular cadherin (EC) repeats or so-called linker regions. These binding sites have varying affinity for calcium occupancy, and different calcium configurations at a linker region increase the mechanical rigidity of the tip link to varying degrees. Here we examine the mechanical strength of cadherin-23 repeats across an ensemble of calcium occupancy states at sites labeled as site1, site2, and site3 (ordered by increasing Kd). We do this through a combination of all-atom steered molecular dynamics simulations and by constructing probability distributions for a configuration ensemble which we analyze by Markov chain Monte Carlo. Simulations predict an increase in the tip link's mechanical strength according to an increase in the number of occupied calcium-binding sites. Surprisingly, our analyses also reveal that for the range of cochlear calcium concentration (20 to 50 uM), the most likely configuration involved a single calcium ion at site3, followed by two calcium ions at site2 and site3, then three calcium ions across all sites, and lastly an empty linker region. We discuss these results in the context of tip-link mediated inner ear mechanotransduction.

Molecular mechanisms underlying CIB function in inner-ear mechanotransduction

In inner-ear mechanotransduction, forces from sound and head movement are converted into nerve impulses to facilitate hearing and balance perception. At the apical surface of inner-ear sensory hair cells are actin-filled protrusions called stereocilia. Successively ascending rows of stereocilia form hair-cell bundles that are deflected by force to open transduction channels for cation influx. This causes hair-cell depolarization and initiates sensory perception. Transmembrane channel-like proteins (TMCs) 1 and 2 have been established as the pore-forming components of hair-cell transduction channels.

Exploring the Structural Elements Responsible for Cis-Homodimerization of Inner Ear Cadherin-23

Cadherin-23 (CDH23) is an enormous protein essential for hearing, balance, and proper eyesight. There are over 100 mutations in CDH23 that affect these processes with varying severity, some leading to deafness, balance disorders, and progressive blindness (Usher Syndrome). In the inner ear, CDH23 makes up the upper half of a proteinaceous filament known as the tip link, which is essential for hearing. Upon stimulation by sound or head movements, the tip link is stretched and conveys force to open the ion channels in the inner ear, thereby leading to the conversion of vibrational stimulus into electrical signals interpreted by the brain as sound. CDH23 is a non-classical cadherin with 27 extracellular cadherin (EC) repeats and a membrane adjacent domain (MAD28). The EC repeats are connected by a linker region containing highly conserved residues that bind calcium ions essential for tip-link function. Electron microscopy images suggest that CDH23 exists as a cis-homodimer within the tip link, however, the structural elements mediating this dimerization are not well determined. To better understand inner-ear mechanotransduction at the molecular level, we have solved high- resolution X-ray crystal structures of 18 CDH23 EC repeats along with 13 of the 26 EC linker regions (Jaiganesh et al., 2018). Here, we present several biochemical experiments that suggest potential sites of parallel dimerization on the extracellular domain of CDH23. Additionally, we present structures of various fragments of CDH23 allowing for closer analysis of deafness causing mutations. These results provide information on the cis-homodimerization of CDH23 and provide deeper insights about how mutations can result in inherited deafness.

Zooming in on Cadherin-23: Structural Diversity and Potential Mechanisms of Inherited Deafness

Cadherin-23 (CDH23) is an essential component of hair-cell tip links, fine filaments that mediate inner-ear mechanotransduction. The extracellular domain of CDH23 forms about three-fourths of the tip link with 27 extracellular cadherin (EC) repeats that are structurally similar but not identical to each other. Calcium (Ca2+) coordination at the EC linker regions is key for tip-link elasticity and function. There are ∼116 sites in CDH23 affected by deafness-causing mutations, many of which alter conserved Ca2+-binding residues. Here we present crystal structures showing 18 CDH23 EC repeats, including the most and least conserved, a fragment carrying disease mutations, and EC repeats with non-canonical Ca2+-binding motif sequences and unusual secondary structure. Complementary experiments show deafness mutations' effects on stability and affinity for Ca2+. Additionally, a model of nine contiguous CDH23 EC repeats reveals helicity and potential parallel dimerization faces. Overall, our studies provide detailed structural insight into CDH23 function in mechanotransduction.

Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins.

Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (KD) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (Tm) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These Tm shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the Tm shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (KD from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.

Beyond Cell-Cell Adhesion: Sensational Cadherins for Hearing and Balance.

Cadherins form a large family of proteins often involved in calcium-dependent cellular adhesion. Although classical members of the family can provide a physical bond between cells, a subset of special cadherins use their extracellular domains to interlink apical specializations of single epithelial sensory cells. Two of these cadherins, cadherin-23 (CDH23) and protocadherin-15 (PCDH15), form extracellular "tip link" filaments that connect apical bundles of stereocilia on hair cells essential for inner-ear mechanotransduction. As these bundles deflect in response to mechanical stimuli from sound or head movements, tip links gate hair-cell mechanosensitive channels to initiate sensory perception. Here, we review the unusual and diverse structural properties of these tip-link cadherins and the functional significance of their deafness-related missense mutations. Based on the structural features of CDH23 and PCDH15, we discuss the elasticity of tip links and models that bridge the gap between the nanomechanics of cadherins and the micromechanics of hair-cell bundles during inner-ear mechanotransduction.

An elastic element in the protocadherin-15 tip link of the inner ear.

Tip link filaments convey force and gate inner-ear hair-cell transduction channels to mediate perception of sound and head movements. Cadherin-23 and protocadherin-15 form tip links through a calcium-dependent interaction of their extracellular domains made of multiple extracellular cadherin (EC) repeats. These repeats are structurally similar, but not identical in sequence, often featuring linkers with conserved calcium-binding sites that confer mechanical strength to them. Here we present the X-ray crystal structures of human protocadherin-15 EC8–EC10 and mouse EC9–EC10, which show an EC8–9 canonical-like calcium-binding linker, and an EC9–10 calcium-free linker that alters the linear arrangement of EC repeats. Molecular dynamics simulations and small-angle X-ray scattering experiments support this non-linear conformation. Simulations also suggest that unbending of EC9–10 confers some elasticity to otherwise rigid tip links. The new structure provides a first view of protocadherin-15's non-canonical EC linkers and suggests how they may function in inner-ear mechanotransduction, with implications for other cadherins.

Steered Molecular Dynamics Simulations of Inner-Ear Cadherins using the Drude Polarizable Force Field

Steered molecular dynamics (SMD) simulations have provided invaluable insights into the unbinding and unfolding mechanisms of proteins with mechanical function. In the case of tip link cadherins involved in inner-ear mechanotransduction, SMD simulations have revealed possible unbinding and unfolding pathways, showed how structural elements and calcium ions are essential for rigidity and unfolding strength, and predicted unbinding and unfolding forces that are relevant at the physiological, fast time scales of sound transduction. Yet all these simulations have used classical force fields that do not take into account atomic polarizability, which is critical for the accurate description of interactions between divalent ions and charged protein side chains. Here, we present SMD simulations of the calcium-dependent cadherin-23 and protocadherin-15 interaction that test the effect of atomic polarizability on its dynamics and mechanical strength. We compared parallel simulations that used either the fixed-atomic charge additive CHARMM36 or the Drude polarizable force fields. A variety of pulling speeds and simulation protocols were tested as well. These simulations shed light on the importance of polarizability on the calcium-dependent mechanics of tip link cadherins.

Using Inner-Ear Cadherins to Test High-Throughput Thermal Screening Assays for Protein-Protein Interactions

High-throughput thermal screening (HTTS) has generally been used to analyze the stability of libraries of mutant proteins, as well as stability changes for proteins with a battery of ligands or a plethora of buffer conditions. In all cases, an environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature shifts under various conditions. HTTS has also been used to characterize protein-protein interactions, but its general applicability to protein complexes remains to be tested. Here, we use the complex formed by the N-terminal fragments of Protocadherin-15 (pcdh15) and Cadherin-23 (cdh23), essential for inner-ear mechanotransduction, to test modified HTTS assays that evaluate protein-protein binding affinities. By mixing cdh23 and pcdh15 at different ratios, we observed increased thermal stability of pcdh15 caused by shifts in chemical equilibrium towards the complex. In addition, protein fragments carrying missense mutations known to cause deafness and reduce binding affinity lack these shifts. These results suggest that HTTS can be used to qualitatively evaluate cadherin protein-protein interactions. We further validated our HTTS results using surface plasmon resonance-based affinity measurements. Surprisingly, we found that in some cases thermal stability shifts correlate well with koff rather than Kd. Our modified protocols might be applicable to any heterodimeric protein complex with affinities ranging from ∼ 1 nanoM to 100 microM.

Single-Molecule Force-Spectroscopy of Inner Ear Proteins

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature allows us to identify single-molecule rupture events in pulling experiments.

Structures and Simulated Dynamics of a Force-Conveying Cadherin Bond Essential for Inner-Ear Mechanotransduction

The senses of hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed directly to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins: protocadherin-15 and cadherin-23. These two proteins are required for mechanotransduction, they are products of deafness genes, and they feature long extracellular domains that are thought to interact tip-to-tip in a calcium-dependent manner. However, the molecular architecture of the complex is unknown and recent studies show that binding between them cannot be mediated by a classical cadherin interface. Moreover, the strength of this complex has not been characterized and the molecular mechanisms leading to cadherin-related deafness remain unexplored.Here we combine X-ray crystallography, microsecond-long molecular dynamics simulations, and binding experiments to characterize the cadherin-23/protocadherin-15 bond. We find a unique cadherin interaction mechanism, with the two most N-terminal cadherin repeats (EC1+2) of each protein interacting to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex becomes unstable upon calcium removal due to increased flexure (entropic stress) of calcium-free cadherin repeats. Finally, we use structures and biochemical measurements to understand molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity, and cancer.

Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals1. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links2,3, fine filaments formed by two atypical cadherins: protocadherin-15 and cadherin-234,5. These two proteins are products of deafness genes6–10 and feature long extracellular domains that interact tip-to-tip5,11 in a Ca2+-dependent manner. However, the molecular architecture of the complex is unknown. Here we combine crystallography, molecular dynamics simulations, and binding experiments to characterize the cadherin-23 and protocadherin-15 bond. We find a unique cadherin interaction mechanism, with the two most N-terminal cadherin repeats (EC1+2) of each protein interacting to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex becomes unstable upon Ca2+ removal due to increased flexure of Ca2+-free cadherin repeats. Finally, we use structures and biochemical measurements to understand molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis12,13, neural connectivity14, and cancer15.