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. 2012 Dec 6;492(7427):128-32.
doi: 10.1038/nature11590. Epub 2012 Nov 7.

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

Marcos Sotomayor  1 , Wilhelm A Weihofen, Rachelle Gaudet, David P Corey
Affiliations

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

Marcos Sotomayor et al. Nature. .

Abstract

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 to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact 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 is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the 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.

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Figures

Figure 1
Figure 1
Structure of tip-link protocadherin-15 bound to cadherin-23. a, Hair-cell stereocilia bundle. A tip-link filament extends from the tip of each stereocilium to the side of its tallest neighbour. b, The tip link formed by a protocadherin-15 parallel dimer interacting tip-to-tip with a cadherin-23 parallel dimer. These proteins feature 11 and 27 extracellular cadherin (EC) repeats, respectively. Inset shows possible arrangement at the junction. c, Ribbon diagram of protocadherin-15 EC1+2 (pcdh-15; purple) bound to cadherin-23 EC1+2 (cdh-23; blue) with Ca2+ ions as green spheres. Arrowheads indicate pcdh-15’s RGGPP loop and cdh-23’s 310 helix. Residues R113, C11, and C99 of pcdh-15 are shown in stick representation. d, Detail of disulfide bond C11-C99 and isoform-dependent residues D4-Y8 at the pcdh-15 N-terminus. e, Detail of Ca2+-binding sites 1, 2, and 3 at the pcdh-15 linker. Protein backbone and sidechains are in cartoon and stick representations, respectively. f, Surface representation of pcdh-15 (purple and pink) and cdh-23 (blue and cyan) as in (c). g, Pcdh-15 and cdh-23 interaction surfaces exposed with interfacing residues labeled.
Figure 2
Figure 2
Pcdh-15+cdh-23 complex formation probed using isothermal titration calorimetry (ITC) and site-directed mutagenesis. a, Raw power vs. time data for pcdh-15 (111 μM) titrated with cdh-23 (1.1 mM) at 10°C (black, WT-WT). Inset shows raw data (blue) for pcdh-15I22A (114 μM) titrated with cdh-23L145G (1.2 mM). b–c, Change in molar enthalpy for pcdh-15 titrated with cdh-23 (black); pcdh-15I22A with cdh-23 (light green); pcdh-15 with cdh-23L145G (dark green); pcdh-15I22A with cdh-23L145G (blue); pcdh-15 with cdh-23S47P (violet); pcdh-15R113G with cdh-23 (magenta); and pcdh-15 with cdh-23D101G (indigo; concentrations in Supplementary Fig. 8). Sigmoidal isothermals were observed only for pcdh-15+cdh-23 and pcdh-15+cdh-23S47P. d–h, Details of pcdh-15+cdh-23 interface, highlighting residue L145 (e), the RXGPP loop (f), and residues I22 (g) and R113 (h). Panel (h) is a 180°-rotated version of panel (g). Protein backbone and interfacing residues (as identified by PISA) are in purple/pink for pcdh-15 and blue/cyan for cdh-23.
Figure 3
Figure 3
Mechanical strength of the pcdh-15+cdh-23 complex probed by steered molecular dynamics (SMD) simulations. a, Snapshots of pcdh-15 (purple) and cdh-23 (blue) unbinding during simulation SNA7 (Supplementary Table 3). The complex is shown in both cartoon and surface representations at the beginning, and in surface representation at indicated time points. Force was applied to the C-termini of both protomers (Supplementary Movies I&II). Green arrows point to broken interfaces. b, Region of gray box in panel (a), showing interacting residues during unbinding. c, Force applied to one C-terminus versus distance between C-termini ends of pcdh-15 and cdh-23. Different traces correspond to independent simulations performed at stretching speeds of 10 (blue and black), 1 (light and dark green), 0.1 (cyan, 1-ns running average shown in black), and 0.02 nm/ns (magenta, 1-ns running average). Snapshots in (a) are indicated by arrowheads. d, Maximum force-peak values vs. stretching speed for unbinding simulations of pcdh-15+cdh-23 started after a 1-ns or 1-μs equilibration (light green, SN2 to SN6; dark green, SNA2 to SNA7; cyan, SN10 to SN13). Simulations SN2-SN6 and SNA2-SNA7 used the S1b structure and SN10-SN13 used S1a; unbinding forces for all three sets were equivalent.
Figure 4
Figure 4
Pcdh-15+cdh-23 complex formation, its Ca2+-dependence, and the role of the deafness mutation R113G, probed using analytical size exclusion chromatography (SEC). Individual traces represent independent experiments. a, top, SEC traces for pcdh-15 and cdh-23 with Ca2+ (red and blue) or with 5 mM EGTA (purple and cyan). A shift upon Ca2+ removal by EGTA was observed for pcdh-15 (purple vs. red curves). middle, SEC traces for pcdh-15+cdh-23 in the presence of Ca2+ (light green) or 5 mM EGTA (dark green). The summation of a purple and a cyan curve from above is shown as a dashed line. The EGTA-treated complex behaved as the sum of its EGTA-treated components, indicating Ca2+-dependent complex formation. bottom, Coomassie-stained SDS-PAGE of eluted fractions from EGTA-treated proteins. b, top, SEC traces for mutant pcdh-15R113G alone (maroon), and mixed with cdh-23 (orange). Wild-type proteins from (a) are shown for comparison. bottom, Coomassie-stained SDS-PAGE of eluted fractions aligned to chromatogram. A reproducible shift in elution volume was observed for the wild-type (green) but not for the mutant mixture (pcdh-15R113G+cdh-23; orange). The shifted peak (1.61 ml) contained both proteins (1.56 to 1.64 ml fractions).
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