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

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.


Related in: MedlinePlus

Constant-velocity SMD simulations of PCDH15 EC8–10.(a) Force applied to N terminus versus end-to-end distance for constant-velocity stretching of PCDH15 EC8–10 at 10 nm ns−1 (simulation S5d, black), 5 nm ns−1 (S5c, turquoise), 1 nm ns−1 (S5a1, light blue), 0.1 nm ns−1 (S5b, 1 ns running average, light green) and 0.02 nm ns−1 (S5e, 1 ns running average, dark green). (b) Snapshots of initial conformation and mechanically induced unbending and unfolding of PCDH15 EC8–10 taken from the 0.02 nm ns−1 simulation (S5e; Movie 2). Protein is shown in ribbon representation and coloured as in Fig. 3. Springs indicate position and direction of the applied forces. (c–f) Force applied to PCDH15 EC8–10 N terminus (S5e, dark green) along with interatomic distances for (c,d) Arg 1,013:O–Ala 1,040:N (magenta) and Arg 1,013:N–Ala 1,040:O (purple); Tyr 1,019:N–Lys 1,108:O (light magenta); and Tyr 1,019:O–Tyr 1,110:N (pink). Rupture of these interactions correlates with unfolding force peaks. (e) Interatomic distances for His 1,007:O–Glu 1,010:N (red), Gly 1,009:O–Ile 1,042:N (orange), (f) Leu 1,004:Cγ–Leu 1,098:Cγ (maroon), and Leu 1,004:Cγ–Ala 1,096Cβ (dark gray). Rupture of these interactions correlates with unbending. A 1 ns running average is shown in all cases. (g–j) Snapshots of the EC9–10 linker during S5e at time points indicated in e,f.
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f7: Constant-velocity SMD simulations of PCDH15 EC8–10.(a) Force applied to N terminus versus end-to-end distance for constant-velocity stretching of PCDH15 EC8–10 at 10 nm ns−1 (simulation S5d, black), 5 nm ns−1 (S5c, turquoise), 1 nm ns−1 (S5a1, light blue), 0.1 nm ns−1 (S5b, 1 ns running average, light green) and 0.02 nm ns−1 (S5e, 1 ns running average, dark green). (b) Snapshots of initial conformation and mechanically induced unbending and unfolding of PCDH15 EC8–10 taken from the 0.02 nm ns−1 simulation (S5e; Movie 2). Protein is shown in ribbon representation and coloured as in Fig. 3. Springs indicate position and direction of the applied forces. (c–f) Force applied to PCDH15 EC8–10 N terminus (S5e, dark green) along with interatomic distances for (c,d) Arg 1,013:O–Ala 1,040:N (magenta) and Arg 1,013:N–Ala 1,040:O (purple); Tyr 1,019:N–Lys 1,108:O (light magenta); and Tyr 1,019:O–Tyr 1,110:N (pink). Rupture of these interactions correlates with unfolding force peaks. (e) Interatomic distances for His 1,007:O–Glu 1,010:N (red), Gly 1,009:O–Ile 1,042:N (orange), (f) Leu 1,004:Cγ–Leu 1,098:Cγ (maroon), and Leu 1,004:Cγ–Ala 1,096Cβ (dark gray). Rupture of these interactions correlates with unbending. A 1 ns running average is shown in all cases. (g–j) Snapshots of the EC9–10 linker during S5e at time points indicated in e,f.

Mentions: To further explore the elasticity of PCDH15 EC8–10, we carried out constant-velocity SMD simulations in which both protein ends are attached to springs that are pulled in opposite directions. The applied force throughout the simulation is obtained from each spring's extension. The PCDH15 EC8–10 equilibrated native state was stretched at speeds that ranged from 0.02 to 10 nm ns−1 (simulations S2a–d, S5a–e, S6a–c, S8a–c; Table 2; Supplementary Movie 2). At fast pulling speeds (5 and 10 nm ns−1), force increased rapidly (Fig. 7a), with unbending quickly followed by unfolding of EC10 at the peak force. For the slower stretching speeds, force barely increased during unbending and straightening of the EC9–10 linker (phase I), with the structure lengthening by ∼4 nm before forces started to increase rapidly (phase II) until unfolding occurred (Fig. 7a–f; Supplementary Fig. 12a). The observation of these two phases suggests that unbending is important for the elastic response of PCDH15.


An elastic element in the protocadherin-15 tip link of the inner ear
Constant-velocity SMD simulations of PCDH15 EC8–10.(a) Force applied to N terminus versus end-to-end distance for constant-velocity stretching of PCDH15 EC8–10 at 10 nm ns−1 (simulation S5d, black), 5 nm ns−1 (S5c, turquoise), 1 nm ns−1 (S5a1, light blue), 0.1 nm ns−1 (S5b, 1 ns running average, light green) and 0.02 nm ns−1 (S5e, 1 ns running average, dark green). (b) Snapshots of initial conformation and mechanically induced unbending and unfolding of PCDH15 EC8–10 taken from the 0.02 nm ns−1 simulation (S5e; Movie 2). Protein is shown in ribbon representation and coloured as in Fig. 3. Springs indicate position and direction of the applied forces. (c–f) Force applied to PCDH15 EC8–10 N terminus (S5e, dark green) along with interatomic distances for (c,d) Arg 1,013:O–Ala 1,040:N (magenta) and Arg 1,013:N–Ala 1,040:O (purple); Tyr 1,019:N–Lys 1,108:O (light magenta); and Tyr 1,019:O–Tyr 1,110:N (pink). Rupture of these interactions correlates with unfolding force peaks. (e) Interatomic distances for His 1,007:O–Glu 1,010:N (red), Gly 1,009:O–Ile 1,042:N (orange), (f) Leu 1,004:Cγ–Leu 1,098:Cγ (maroon), and Leu 1,004:Cγ–Ala 1,096Cβ (dark gray). Rupture of these interactions correlates with unbending. A 1 ns running average is shown in all cases. (g–j) Snapshots of the EC9–10 linker during S5e at time points indicated in e,f.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120219&req=5

f7: Constant-velocity SMD simulations of PCDH15 EC8–10.(a) Force applied to N terminus versus end-to-end distance for constant-velocity stretching of PCDH15 EC8–10 at 10 nm ns−1 (simulation S5d, black), 5 nm ns−1 (S5c, turquoise), 1 nm ns−1 (S5a1, light blue), 0.1 nm ns−1 (S5b, 1 ns running average, light green) and 0.02 nm ns−1 (S5e, 1 ns running average, dark green). (b) Snapshots of initial conformation and mechanically induced unbending and unfolding of PCDH15 EC8–10 taken from the 0.02 nm ns−1 simulation (S5e; Movie 2). Protein is shown in ribbon representation and coloured as in Fig. 3. Springs indicate position and direction of the applied forces. (c–f) Force applied to PCDH15 EC8–10 N terminus (S5e, dark green) along with interatomic distances for (c,d) Arg 1,013:O–Ala 1,040:N (magenta) and Arg 1,013:N–Ala 1,040:O (purple); Tyr 1,019:N–Lys 1,108:O (light magenta); and Tyr 1,019:O–Tyr 1,110:N (pink). Rupture of these interactions correlates with unfolding force peaks. (e) Interatomic distances for His 1,007:O–Glu 1,010:N (red), Gly 1,009:O–Ile 1,042:N (orange), (f) Leu 1,004:Cγ–Leu 1,098:Cγ (maroon), and Leu 1,004:Cγ–Ala 1,096Cβ (dark gray). Rupture of these interactions correlates with unbending. A 1 ns running average is shown in all cases. (g–j) Snapshots of the EC9–10 linker during S5e at time points indicated in e,f.
Mentions: To further explore the elasticity of PCDH15 EC8–10, we carried out constant-velocity SMD simulations in which both protein ends are attached to springs that are pulled in opposite directions. The applied force throughout the simulation is obtained from each spring's extension. The PCDH15 EC8–10 equilibrated native state was stretched at speeds that ranged from 0.02 to 10 nm ns−1 (simulations S2a–d, S5a–e, S6a–c, S8a–c; Table 2; Supplementary Movie 2). At fast pulling speeds (5 and 10 nm ns−1), force increased rapidly (Fig. 7a), with unbending quickly followed by unfolding of EC10 at the peak force. For the slower stretching speeds, force barely increased during unbending and straightening of the EC9–10 linker (phase I), with the structure lengthening by ∼4 nm before forces started to increase rapidly (phase II) until unfolding occurred (Fig. 7a–f; Supplementary Fig. 12a). The observation of these two phases suggests that unbending is important for the elastic response of PCDH15.

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.


Related in: MedlinePlus