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

Nature volume 492, pages 128–132 (2012)Cite this article

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Abstract

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 known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness6,7,8,9,10 and feature long extracellular domains that interact tip-to-tip5,11 in a Ca2+-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 Ca2+ removal owing to increased flexure of Ca2+-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 morphogenesis12,13, neural connectivity14 and cancer15.

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Figure 1: Structure of tip-link protocadherin 15 bound to cadherin 23.
Figure 2: Pcdh15-EC1+2–Cdh23-EC1+2 complex formation probed using ITC and site-directed mutagenesis.
Figure 3: Mechanical strength of the Pcdh15-EC1+2–Cdh23-EC1+2 complex probed by SMD simulations.
Figure 4: Pcdh15-EC1+2–Cdh23-EC1+2 complex formation, its Ca 2+ dependence and the role of the deafness mutation R113G, probed using analytical SEC.

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Protein Data Bank

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 4apx (S1a), 4axw (S1b), 4aq8 (S2), 4aqa (S3) and 4aqe (S4).

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Acknowledgements

We thank B. Derfler for assistance with mutagenesis, V. D’Souza and her laboratory for advice on calorimetry, and the Corey and Gaudet laboratories for discussions. Full-length complementary DNAs of Cdh23 and Pcdh15 used as template for some of our constructs were provided by U. Müller (The Scripps Research Institute) and T. B. Friedman (National Institutes of Health (NIH)). This work was supported by the NIH (R01 DC02281 to D.P.C.; RC2GM093307 to National Resource for Biomedical Supercomputing/Pittsburgh Supercomputing Center) and the National Science Foundation through TeraGrid/XSEDE (TRAC MCB080015). Simulations were performed at the NCSA-Abe, NICS-Kraken, TACC-Ranger and PSC-Anton supercomputers. Use of Advanced Photon Source beamlines was supported by NIH award RR-15301 and Department of Energy (DOE) contract no. DE-AC02-06CH11357. Use of Advanced Light Source beamline 4.2.2 was supported by DOE contract no. DE-AC02-05CH11231. M.S. was a Howard Hughes Medical Institute (HHMI) Fellow of the Helen Hay Whitney Foundation and D.P.C. is an HHMI Investigator.

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Author notes

  1. Wilhelm A. Weihofen

    Present address: Present address: Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Marcos Sotomayor & David P. Corey

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, 02138, Massachusetts, USA

    Wilhelm A. Weihofen & Rachelle Gaudet

Authors
  1. Marcos Sotomayor
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Contributions

All authors participated in all parts of this study. M.S. did all experiments and simulations.

Corresponding authors

Correspondence to Rachelle Gaudet or David P. Corey.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains the Supplementary Discussion, Supplementary Tables 1-3, Supplementary Figures 1-19 and additional references. (PDF 9411 kb)

Forced unbinding of complex

Forced unbinding of Pcdh15-EC1+2−Cdh23-EC1+2 (simulation SNA7; trajectory shown from t = 270 ns up to t = 336 ns). Protein is depicted in cartoon representation, with Pcdh15-EC1+2 in purple, Cdh23-EC1+2 in blue, and Ca2+ in green. C-terminal Cα atoms are red. Molecular surfaces for Pcdh15-EC1+2 and Cdh23-EC1+2 are shown in transparent purple and blue. Version B shows the same trajectory with opaque molecular surfaces. (MOV 4431 kb)

Details of complex forced unbinding

Details of Pcdh15-EC1+2−Cdh23-EC1+2 forced unbinding (simulation SNA7; trajectory shown from t = 270 ns up to t = 336 ns). Protein is depicted as in Video I, with residues at the interface shown in stick representation. Molecular surfaces are not shown in version A. Version B shows opaque and transparent surfaces for Pcdh15-EC1+2 and Cdh23-EC1+2, respectively. (MOV 6319 kb)

Equilibration of complex in the presence of Ca2+

Equilibration of Pcdh15-EC1+2−Cdh23-EC1+2 in the presence of Ca2+ (simulation SA1, lasting 1 µs). Protein is depicted in cartoon representation. Pcdh15-EC1+2 is in purple, Cdh23-EC1+2 in blue, and Ca2+ in green. (MPG 3033 kb)

Equilibration of complex in the absence of Ca2+

Equilibration of Pcdh15-EC1+2−Cdh23-EC1+2 in the absence of Ca2+ (simulation SA3, lasting 1 µs). Protein is depicted in cartoon representation and coloured as in Video 3. (MPG 3072 kb)

Dynamics of contacts during equilibrium simulations

Dynamics of contacts between Pcdh15-EC1+2 and Cdh23-EC1+2 during equilibrium MD simulations. Contact maps between residue pairs involved in the Pcdh15-EC1+2−Cdh23-EC1+2 interface are shown throughout microsecond-long MD simulations performed in the NpT ensemble with and without Ca2+ (SA1 left; SA3 right). The distance between pairs of Cα atoms is displayed using a linear gray scale (0 Å: black; > 10 Å: white). A red box highlights location of native contacts lost at the end of simulation SA3 without Ca2+. (MPG 3533 kb)

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Sotomayor, M., Weihofen, W., Gaudet, R. et al. Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction. Nature 492, 128–132 (2012). https://doi.org/10.1038/nature11590

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