Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 18;115(12):2368-2385.
doi: 10.1016/j.bpj.2018.11.010. Epub 2018 Nov 16.

A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

Pedro De-la-Torre  1 , Deepanshu Choudhary  1 , Raul Araya-Secchi  2 , Yoshie Narui  1 , Marcos Sotomayor  3
Affiliations

A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

Pedro De-la-Torre et al. Biophys J. .

Abstract

The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) "repeats" that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of nonclassical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has 11 EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size-exclusion chromatography coupled to multiangle laser light scattering and small-angle x-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the "giant" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

PubMed Disclaimer

Figures

Figure 1
Figure 1
PCDH15 architecture and structure of the EC10-MAD12 fragment. (A) Schematic of the cadherin tip link and the PCDH15 extracellular domain. Inset shows the location of the fragment studied here. (B) Ribbon diagram of ss PCDH15 EC10-MAD12 (magenta and purple) with transparent molecular surface. Ca2+ ions are shown as green spheres. Red arrow indicates β2”-310-β3’ hook. Dashed line indicates unresolved region. Membrane and mechanotransduction channel are indicated (not to scale; TMHS transmembrane helices are purple, TMIE transmembrane helix is orange, TMC1 dimer is light purple). (C) Topology diagram of PCDH15 EC10-MAD12. A typical cadherin fold is observed for EC10 and EC11. A ferredoxin-like fold is observed for MAD12. Intramolecular EC11-MAD12 contact is highlighted by red dashed lines. Blue arrows indicate kinks. Cyan and green circles highlight sites involved in hereditary deafness (causal and correlated mutations, respectively).
Figure 2
Figure 2
Structural details of EC10-MAD12 fragment. (A) Detail of EC10–11 linker region. Ca2+ ions are shown in green and Ca2+-coordinating side chains in yellow sticks. (B) MAD12 in ribbon representation with secondary structure elements labeled. Blue arrows indicate kinks. Inset shows β sheet arrangement. (C and D) A detailed view of the intramolecular interactions at the EC11-MAD12 interface. Residues are shown in yellow sticks and labeled; some backbone atoms are omitted for clarity. Cyan circle indicates site involved in inherited deafness (p.G1130R). Green star indicates the first residue of MAD12. (E) Interaction surface exposed (gray). Red star indicates connection point. Deafness mutation site p.G1130 is highlighted in white. Rotations in (C) and (E) are indicated with respect to view of ss PCDH15 EC10-MAD12 in Fig. 1B. Rotation in (D) is with respect to view in (C).
Figure 3
Figure 3
Dimerization interface in ss PCDH15 EC10-MAD12. (A and B) Front and bottom views of ss PCDH15 EC10-MAD12 dimer in ribbon (magenta and mauve) with a transparent molecular surface. Black-dashed boxes indicate EC10-EC10 interface (A, top) and MAD12’s hook (A, bottom). Dashed blue arrows indicate opening crossed by p.R1137. Solid black arrows indicate binding interface between protomers. (C) Detail of EC10-EC10 interface (black box in A). Protein backbone is shown in ribbons and relevant residues are shown as yellow sticks. (D) Side view of dimer in ribbon (left) and opaque surface (right) representations. (E) Interaction surfaces exposed and shown in gray with interfacing residues labeled. Missing loop in one protomer is indicated by dashed-black line. Deafness associated sites are labeled in green.
Figure 4
Figure 4
PCDH15 EC10-MAD12 forms a parallel dimer in solution. (A) AUC experiments for mouse protein refolded from bacteria (mm, left), mouse protein purified from mammalian cells (mm∗, middle), and pig protein refolded from bacteria (ss, right). Peaks at S > 4 represent dimeric states. (B) X-ray scattering intensity as a function of the scattering vector q (SAXS profile) for pig (magenta, left) and mouse (purple, right) PCDH15 EC10-MAD12. Predicted scattering intensities from the structure (6BXZ) obtained with FoXS are shown in maroon-dashed lines (χ2 = 5.09/3.42), and theoretical scattering curves obtained from ab initio modeling (DAMMIF) and from flexible refinement with SREFLEX are shown in red (χ2 = 1.44/1.14) and blue (χ2 = 1.64/1.23), respectively. (C) Guinier plot of the low q region of the SAXS data for ss and mm (magenta and purple circles, respectively) PCDH15 EC10-MAD12. Magenta and purple solid lines show linear fits from which the gradient of the slope (−Rg2/3) was used to estimate Rg. (D) Real-space pair distribution function P(r) from SAXS data for ss and mm PCDH15 EC10-MAD12 (magenta and purple). (E and F) Superposition of the ss PCDH15 EC10-MAD12 structure (6BXZ) with refined low-resolution bead models obtained for pig (44 ± 3 Å) and mouse (37 ± 3 Å) PCDH15 EC10-MAD12 data.
Figure 5
Figure 5
Constant-velocity SMD simulations of ss PCDH15 EC10-MAD12. (A) Snapshots of monomeric ss PCDH15 EC10-MAD12 stretching from Simulation S1d (0.1 nm/ns; Table S4). Protein is shown in mauve ribbon representation, Ca2+ ions as green spheres, and stretched terminal Cα atoms as red spheres. Springs indicate position and direction of applied forces. Red bar illustrates direction of PCDH15’s MAD12 Hook. (B) Force applied to C terminus versus end-to-end distance for constant velocity stretching of monomeric ss PCDH15 EC10-MAD12 at 10 nm/ns (S1b, blue), 1 nm/ns (S1c, light green), 0.1 nm/ns (S1d, dark green; 1-ns running average shown in black) and 0.02 nm/ns (S1e, 1-ns running average shown in magenta). Red arrowheads indicate time-points for S1d illustrated in (A). (C) Snapshots of dimeric ss PCDH15 EC10-MAD12 stretching from simulation S2d as in (A), with subunits in magenta and mauve. (D) Force applied to N- and C-termini of one subunit (mauve in C) versus end-to-end distance for constant-velocity stretching of dimeric ss PCDH15 EC10-MAD12 at 10 nm/ns (S2b, blue and indigo), 1 nm/ns (S2c, light green and cyan), and 0.1 nm/ns (S2d, dark green and turquoise; 1-ns running average shown in black and maroon). Red arrowheads indicate time points illustrated in (C). (E) Snapshots of dimeric ss PCDH15 EC10-MAD12 stretching from simulation S2g as in (C). Stretching was carried out by attaching two slabs to springs that were in turn attached to the terminal ends of each protein. Slabs were moved in opposite directions through individual springs. (F) Force applied to each of the slabs versus slab separation for constant-velocity stretching of dimeric ss PCDH15 EC10-MAD12 at 10 nm/ns (S2e, blue and indigo), 1 nm/ns (S2f, light green and cyan), 0.1 nm/ns (S2g, dark green and turquoise; 1-ns running average shown in black and maroon), and 0.02 nm/ns (S2h, 1-ns running average shown in magenta and violet). (G) In silico force peak maxima versus stretching speed for CDH23 EC1-2 unfolding (red, maroon, yellow, and orange) (103), for CDH23 EC1-2 and PCDH15 EC1-2 handshake unbinding (cyan-4AQ8, dark green-4AXW, and light green-4AXW∗ [after 1 μs-long equilibration]) (43, 112), and for MAD12 unfolding (magenta-6BXZ for simulations S1b–e; purple-6BXZ∗ for averages obtained for simulations S2b–d). (H) In silico force peak maxima versus stretching speed for unbinding of pairs of CDH23 EC1-2 and PCDH15 EC1-2 handshakes (blue) (43) and for unfolding of MAD12 using slabs (magenta, simulations S2e–h).
Figure 6
Figure 6
Presence of putative MADs in the cadherin superfamily. (A) Domain organization of human cadherins predicted to feature domains similar to PCDH15 MAD12. FAT cadherins are grouped according to post-MAD segment architecture. Preceding EC repeats are not labeled for FAT1 (EC34) and FAT3 (EC32). (B) Structural comparison between xl Nup54 αβ domain (5C2U) (98) and ss PCDH15 MAD12 (PDB: 6BXZ; front and backviews). Molecules are colored according to structural homology per residue (Qres) (117). Blue color implies high structural conservation, whereas red implies poor structural conservations. Beginning of β1 and α3 were truncated out for better structural alignment. (C–E) Structural models of MADs from atypical cadherins (hs PCDH15 MAD12; hs CDH23 MAD28; hs PCDH24 MAD10) superposed on the crystal structure of ss PCDH15 MAD12 (6BXZ) are shown, as in (B).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hulpiau P., van Roy F. Molecular evolution of the cadherin superfamily. Int. J. Biochem. Cell Biol. 2009;41:349–369. - PubMed
    1. Brasch J., Harrison O.J., Shapiro L. Thinking outside the cell: how cadherins drive adhesion. Trends Cell Biol. 2012;22:299–310. - PMC - PubMed
    1. Hirano S., Takeichi M. Cadherins in brain morphogenesis and wiring. Physiol. Rev. 2012;92:597–634. - PubMed
    1. Sotomayor M., Gaudet R., Corey D.P. Sorting out a promiscuous superfamily: towards cadherin connectomics. Trends Cell Biol. 2014;24:524–536. - PMC - PubMed
    1. Ringwald M., Schuh R., Mayer S. The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion. EMBO J. 1987;6:3647–3653. - PMC - PubMed

Publication types

LinkOut - more resources