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

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ABSTRACT

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.

No MeSH data available.


Structure of PCDH15 EC9–10 interface.(a) Surface representation of PCDH15 EC8–10 (magenta). The EC9–10 interface is shown with its EC9–10 linker (violet), a hydrophobic core (dark pink) and supporting residues (purple). (b) PCDH15 EC9–10 interaction surfaces exposed (left and middle panels) and coloured as in a with interfacing residues labeled. Right panel shows side view as in a. Red spheres correspond to a pair of crystallographic water molecules. (c) Conservation of residues in the EC9–10 interface according to ConSurf80 and the alignment in Supplementary Fig. 4. (d,e) Detail of the EC9–10 interface. Protein backbone is shown as ribbons and relevant residues are shown as sticks. Some backbone atoms are omitted for clarity. (f) Detail of backbone-hydrogen bonds (dashed lines) found in the EC9–10 linker.
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f3: Structure of PCDH15 EC9–10 interface.(a) Surface representation of PCDH15 EC8–10 (magenta). The EC9–10 interface is shown with its EC9–10 linker (violet), a hydrophobic core (dark pink) and supporting residues (purple). (b) PCDH15 EC9–10 interaction surfaces exposed (left and middle panels) and coloured as in a with interfacing residues labeled. Right panel shows side view as in a. Red spheres correspond to a pair of crystallographic water molecules. (c) Conservation of residues in the EC9–10 interface according to ConSurf80 and the alignment in Supplementary Fig. 4. (d,e) Detail of the EC9–10 interface. Protein backbone is shown as ribbons and relevant residues are shown as sticks. Some backbone atoms are omitted for clarity. (f) Detail of backbone-hydrogen bonds (dashed lines) found in the EC9–10 linker.

Mentions: The PCDH15 EC9–10 interface can be divided into three components (Fig. 3a,b). The first one is the EC9–10 linker formed by residues Val 1,005 to Arg 1,013, which includes the EC9–10 310 helix. The second corresponds to a mostly hydrophobic core formed by residues Met 913, Thr 978, Ile 980 and Leu 1,004 in EC9 interacting with residues Val 1,093, Leu 1,098 and Val 1,100 in the EC10 FG-α loop. Residues Leu 1,006 and Ile 1,011, which are part of the EC9–10 linker, also participate in hydrophobic core interactions. The third component of the interface consists of two supporting loops (Val 914–Asp 917 in EC9 and Ala 1,040–Ser 1,046 in EC10) that may provide stability to the linker but do not form part of the interface between EC9 and EC10. Conservation analysis of the interface reveals that most of the hydrophobic residues are highly conserved, while some of the polar and charged residues present in the EC9–10 linker and in the EC10 FG-α loop show more variability (Fig. 3c). The buried surface area of the EC9–10 interface is 389 Å2. A structure of mouse Pcdh15 EC9–10 (without EC8), crystalized in a different condition and space group (Table 1; Supplementary Fig. 7), shows the same unique features. This suggests that the EC9–10 interface is robust and that the observed features are not a crystallographic artifact (Supplementary Discussion).


An elastic element in the protocadherin-15 tip link of the inner ear
Structure of PCDH15 EC9–10 interface.(a) Surface representation of PCDH15 EC8–10 (magenta). The EC9–10 interface is shown with its EC9–10 linker (violet), a hydrophobic core (dark pink) and supporting residues (purple). (b) PCDH15 EC9–10 interaction surfaces exposed (left and middle panels) and coloured as in a with interfacing residues labeled. Right panel shows side view as in a. Red spheres correspond to a pair of crystallographic water molecules. (c) Conservation of residues in the EC9–10 interface according to ConSurf80 and the alignment in Supplementary Fig. 4. (d,e) Detail of the EC9–10 interface. Protein backbone is shown as ribbons and relevant residues are shown as sticks. Some backbone atoms are omitted for clarity. (f) Detail of backbone-hydrogen bonds (dashed lines) found in the EC9–10 linker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120219&req=5

f3: Structure of PCDH15 EC9–10 interface.(a) Surface representation of PCDH15 EC8–10 (magenta). The EC9–10 interface is shown with its EC9–10 linker (violet), a hydrophobic core (dark pink) and supporting residues (purple). (b) PCDH15 EC9–10 interaction surfaces exposed (left and middle panels) and coloured as in a with interfacing residues labeled. Right panel shows side view as in a. Red spheres correspond to a pair of crystallographic water molecules. (c) Conservation of residues in the EC9–10 interface according to ConSurf80 and the alignment in Supplementary Fig. 4. (d,e) Detail of the EC9–10 interface. Protein backbone is shown as ribbons and relevant residues are shown as sticks. Some backbone atoms are omitted for clarity. (f) Detail of backbone-hydrogen bonds (dashed lines) found in the EC9–10 linker.
Mentions: The PCDH15 EC9–10 interface can be divided into three components (Fig. 3a,b). The first one is the EC9–10 linker formed by residues Val 1,005 to Arg 1,013, which includes the EC9–10 310 helix. The second corresponds to a mostly hydrophobic core formed by residues Met 913, Thr 978, Ile 980 and Leu 1,004 in EC9 interacting with residues Val 1,093, Leu 1,098 and Val 1,100 in the EC10 FG-α loop. Residues Leu 1,006 and Ile 1,011, which are part of the EC9–10 linker, also participate in hydrophobic core interactions. The third component of the interface consists of two supporting loops (Val 914–Asp 917 in EC9 and Ala 1,040–Ser 1,046 in EC10) that may provide stability to the linker but do not form part of the interface between EC9 and EC10. Conservation analysis of the interface reveals that most of the hydrophobic residues are highly conserved, while some of the polar and charged residues present in the EC9–10 linker and in the EC10 FG-α loop show more variability (Fig. 3c). The buried surface area of the EC9–10 interface is 389 Å2. A structure of mouse Pcdh15 EC9–10 (without EC8), crystalized in a different condition and space group (Table 1; Supplementary Fig. 7), shows the same unique features. This suggests that the EC9–10 interface is robust and that the observed features are not a crystallographic artifact (Supplementary Discussion).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.