Extracellular Cadherin Repeats Research Articles

CELSR1, a core planar cell polarity protein, features a weakly adhesive and flexible cadherin ectodomain

Planar cell polarity (PCP), essential to multicellular developmental processes, arises when cells polarize and align across tissues. Central to PCP is CELSR1, an atypical cadherin featuring a long ectodomain with nine extracellular cadherin (EC) repeats, a membrane adjacent domain (MAD10), and several characteristic adhesion GPCR domains. Cell-based aggregation assays have demonstrated CELSR1's homophilic adhesive nature, but mechanistic details are missing. Here, we investigate the possible adhesive properties and structures of CELSR1 EC repeats. Our bead aggregation assays do not support strong adhesion by EC repeats alone. Consistently, EC1-4 only dimerizes at high concentration in solution. Crystal structures of human CELSR1 EC1-4 and EC4-7 reveal typical folds and a non-canonical linker between EC5 and EC6. Simulations and experiments using EC4-7 indicate flexibility at EC5-6, and solution experiments show EC7-MAD10-mediated dimerization. Our results suggest weak homophilic adhesion by CELSR1 cadherin repeats and provide mechanistic insights into the structural determinants of CELSR1 function.

Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we use computational and experimental approaches to delineate the binding surface of the hantavirus glycoprotein complex on PCDH1’s first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influences the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this and a neighboring residue substantially protects Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F

Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3–5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.

Structural characterization of mini-PCDH15 proteins for Usher syndrome type 1F therapy.

Hair cells, the sensory cells of the inner ear, carry actin-filled microvillus-like projections called stereocilia, which are interconnected by long protein filaments called “tip links”. Upon sound stimulation, tip links enable hair-cell mechanotransduction, the critical conversion of mechanical deflection into an electrical signal our brain can understand. Tip links are formed by two pairs of large Ca2+-binding proteins, cadherin-23 (CDH23) and protocadherin-15 (PCDH15), interacting tip-to-tip in a Ca2+-dependent manner. The ectodomain of PCDH15 is made of 11 extracellular cadherin (EC) repeats, with EC linkers stabilized by Ca2+ ions bound to acidic residues. Mutations in PCDH15 cause Usher Syndrome Type 1F, characterized by congenital hearing loss, balance deficit, and progressive blindness. Adeno-associated virus (AAV) vectors have been found to be efficient and effective for gene therapy in the inner ear. However, the PCDH15 coding sequence is too large to fit in AAV. We have developed novel “mini-PCDH15” constructs that retain critical domains but lack 3-5 EC repeats, and that consequently fit in AAV vectors. Some of the mini-PCDH15s are functional and rescue hearing and balance in Pcdh15-knockout mice despite having engineered Ca2+-binding linkers that are not found in nature. Here, we present structural and computational studies of mini-PCDH15s, including an X-ray crystal structure of an artificial EC3-EC7 bent linker of a mini-PCDH15 that partially rescues hearing. In addition, we report on biochemical assays, low-resolution cryo-EM, and molecular dynamics simulations used to compare the flexibility, rigidity, and Ca2+-binding ability of wild-type and mini-PCDH15 ectodomains. Our mini-PCDH15s show different stability and behave as dimers in solution, showing diverse conformational arrangements that might modulate binding to CDH23. Our results provide an atomistic view of engineered mini-PCDH15s and promote the use of rational, iterative, structure-based mini-gene approach to develop gene therapies for large proteins.

Calcium-dependent strength of the inner-ear cadherin-23 ectodomain.

Cadherin proteins are essential for multicellular organisms and are involved in multiple processes including calcium-dependent cell-cell adhesion and inner-ear mechanotransduction. Cadherin-23 is an integral component of a fibrous protein filament, called the tip link, that is ultimately responsible for converting the mechanical energy of sound into the opening and closing of a gated ion channel that triggers signaling through the auditory nerve. Proper functioning of the tip link depends on its mechanical strength, which is stabilized by interactions of calcium ions with nearby protein residues at discrete triplet binding sites between extracellular cadherin (EC) repeats or so-called linker regions. These binding sites have varying affinity for calcium occupancy, and different calcium configurations at a linker region increase the mechanical rigidity of the tip link to varying degrees. Here we examine the mechanical strength of cadherin-23 repeats across an ensemble of calcium occupancy states at sites labeled as site1, site2, and site3 (ordered by increasing Kd). We do this through a combination of all-atom steered molecular dynamics simulations and by constructing probability distributions for a configuration ensemble which we analyze by Markov chain Monte Carlo. Simulations predict an increase in the tip link's mechanical strength according to an increase in the number of occupied calcium-binding sites. Surprisingly, our analyses also reveal that for the range of cochlear calcium concentration (20 to 50 uM), the most likely configuration involved a single calcium ion at site3, followed by two calcium ions at site2 and site3, then three calcium ions across all sites, and lastly an empty linker region. We discuss these results in the context of tip-link mediated inner ear mechanotransduction.

Elastic versus brittle mechanical responses predicted for dimeric cadherin complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated α, β, and γ clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function.

Elucidating the structural determinant of CELSR1 function involved in planar cell polarity

Planar cell polarity (PCP) refers to a periodic arrangement of cells across an axis of a tissue plane through asymmetric localization of five core PCP proteins, one of which is CELSR1, an atypical cadherin. In PCP, CELSR1 localizes to both sides of the cell and selectively recruits other core PCP transmembrane proteins, Frizzled3/6 and Vangl2 to opposing sides within the cell. Cadherins, which are primarily known for cell adhesion, depend on their extracellular cadherin (EC) repeats for function.

Extracellular domains of E-cadherin determine key mechanical phenotypes of an epithelium through cell- and non-cell-autonomous outside-in signaling.

Cadherins control intercellular adhesion in most metazoans. In vertebrates, intercellular adhesion differs considerably between cadherins of type-I and type-II, predominantly due to their different extracellular regions. Yet, intercellular adhesion critically depends on actomyosin contractility, in which the role of the cadherin extracellular region is unclear. Here, we dissect the roles of the Extracellular Cadherin (EC) Ig-like domains by expressing chimeric E-cadherin with E-cadherin and cadherin-7 Ig-like domains in cells naturally devoid of cadherins. Using cell-cell separation, cortical tension measurement, tissue stretching and migration assays, we show that distinct EC repeats in the extracellular region of cadherins differentially modulate epithelial sheet integrity, cell-cell separation forces, and cell cortical tension with the Cdc42 pathway, which further differentially regulate epithelial tensile strength, ductility, and ultimately collective migration. Interestingly, dissipative processes rather than static adhesion energy mostly dominate cell-cell separation forces. We provide a framework for the emergence of epithelial phenotypes from cell mechanical properties dependent on EC outside-in signaling.

Mechanism of dimerization and structural features of human LI-cadherin

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.

Crystal structure of the nonclassical cadherin-17 N-terminus and implications for its adhesive binding mechanism.

The cadherin superfamily of calcium-dependent cell-adhesion proteins has over 100 members in the human genome. All members of the superfamily feature at least a pair of extracellular cadherin (EC) repeats with calcium-binding sites in the EC linker region. The EC repeats across family members form distinct complexes that mediate cellular adhesion. For instance, classical cadherins (five EC repeats) strand-swap their N-termini and exchange tryptophan residues in EC1, while the clustered protocadherins (six EC repeats) use an extended antiparallel `forearm handshake' involving repeats EC1-EC4. The 7D-cadherins, cadherin-16 (CDH16) and cadherin-17 (CDH17), are the most similar to classical cadherins and have seven EC repeats, two of which are likely to have arisen from gene duplication of EC1-2 from a classical ancestor. However, CDH16 and CDH17 lack the EC1 tryptophan residue used by classical cadherins to mediate adhesion. The structure of human CDH17 EC1-2 presented here reveals features that are not seen in classical cadherins and that are incompatible with the EC1 strand-swap mechanism for adhesion. Analyses of crystal contacts, predicted glycosylation and disease-related mutations are presented along with sequence alignments suggesting that the novel features in the CDH17 EC1-2 structure are well conserved. These results hint at distinct adhesive properties for 7D-cadherins.

Elucidating the Adhesive Mechanism of the Atypical Cadherin CELSR1 Involved in Planar Cell Polarity

In multicellular organisms, planar cell polarity (PCP) ensures the proper development of diverse organs during embryogenesis. PCP is characterized by the asymmetric distribution of two groups of transmembrane protein complexes on the apical region of the cell: Cadherin EGF like Laminin G Seven pass type G protein Receptor 1 (CELSR1) and Frizzled (Fzd3/6) on one side; CELSR1 and Vang like- (Vang2) on the opposite side. Among these proteins, CELSR1, an atypical cadherin with nine extracellular cadherin (EC) repeats, modulates the transmission of polarization information across the apical plane through homophilic interactions between adjacent cells. We use a combination of biophysical and biochemical techniques such as bead aggregation assays and multi angle light scattering to explore the adhesive function of CELSR1. While CELSR1 has additional extracellular domains such as EGF, LamG and EGF_Lam following the nine EC repeats, we show that adhesion can be mediated by the first few N-terminal EC repeats rather than the whole extracellular domain of the protein. These results illuminate the adhesive mechanism of CELSR1 important for PCP.

Exploring the Structural Elements Responsible for Cis-Homodimerization of Inner Ear Cadherin-23

Cadherin-23 (CDH23) is an enormous protein essential for hearing, balance, and proper eyesight. There are over 100 mutations in CDH23 that affect these processes with varying severity, some leading to deafness, balance disorders, and progressive blindness (Usher Syndrome). In the inner ear, CDH23 makes up the upper half of a proteinaceous filament known as the tip link, which is essential for hearing. Upon stimulation by sound or head movements, the tip link is stretched and conveys force to open the ion channels in the inner ear, thereby leading to the conversion of vibrational stimulus into electrical signals interpreted by the brain as sound. CDH23 is a non-classical cadherin with 27 extracellular cadherin (EC) repeats and a membrane adjacent domain (MAD28). The EC repeats are connected by a linker region containing highly conserved residues that bind calcium ions essential for tip-link function. Electron microscopy images suggest that CDH23 exists as a cis-homodimer within the tip link, however, the structural elements mediating this dimerization are not well determined. To better understand inner-ear mechanotransduction at the molecular level, we have solved high- resolution X-ray crystal structures of 18 CDH23 EC repeats along with 13 of the 26 EC linker regions (Jaiganesh et al., 2018). Here, we present several biochemical experiments that suggest potential sites of parallel dimerization on the extracellular domain of CDH23. Additionally, we present structures of various fragments of CDH23 allowing for closer analysis of deafness causing mutations. These results provide information on the cis-homodimerization of CDH23 and provide deeper insights about how mutations can result in inherited deafness.

Identification of an adhesive interface for the non-clustered \u03b41 protocadherin-1 involved in respiratory diseases

Cadherins form a large family of calcium-dependent adhesive proteins involved in morphogenesis, cell differentiation, and neuronal connectivity. Non-clustered δ1 protocadherins form a cadherin subgroup of proteins with seven extracellular cadherin (EC) repeats and cytoplasmic domains distinct from those of classical cadherins. Non-clustered δ1 protocadherins mediate homophilic adhesion and have been implicated in various diseases including asthma, autism, and cancer. Here we present X-ray crystal structures of human Protocadherin-1 (PCDH1), a δ1-protocadherin member essential for New World Hantavirus infection that is typically expressed in the brain, airway epithelium, skin keratinocytes, and lungs. The structures suggest a binding mode that involves antiparallel overlap of repeats EC1 to EC4. Mutagenesis combined with binding assays and biochemical experiments validated this mode of adhesion. Overall, these results reveal the molecular mechanism underlying adhesiveness of PCDH1 and δ1-protocadherins, also shedding light on PCDH1’s role in maintaining airway epithelial integrity, the loss of which causes respiratory diseases.

FAT4 Fine-Tunes Kidney Development by Regulating RET Signaling

FAT4 mutations lead to several human diseases that disrupt the normal development of the kidney. However, the underlying mechanism remains elusive. In studying the duplex kidney phenotypes observed upon deletion of Fat4 in mice, we have uncovered an interaction between the atypical cadherin FAT4 and RET, a tyrosine kinase receptor essential for kidney development. Analysis of kidney development in Fat4-/- kidneys revealed abnormal ureteric budding and excessive RET signaling. Removal of one copy of the RET ligand Gdnf rescues Fat4-/- kidney development, supporting the proposal that loss of Fat4 hyperactivates RET signaling. Conditional knockout analyses revealed a non-autonomous role for Fat4 in regulating RET signaling. Mechanistically, we found that FAT4 interacts with RET through extracellular cadherin repeats. Importantly, expression of FAT4 perturbs the assembly of the RET-GFRA1-GDNF complex, reducing RET signaling. Thus, FAT4 interacts with RET to fine-tune RET signaling, establishing a juxtacrine mechanism controlling kidney development.

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

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.

Zooming in on Cadherin-23: Structural Diversity and Potential Mechanisms of Inherited Deafness

Cadherin-23 (CDH23) is an essential component of hair-cell tip links, fine filaments that mediate inner-ear mechanotransduction. The extracellular domain of CDH23 forms about three-fourths of the tip link with 27 extracellular cadherin (EC) repeats that are structurally similar but not identical to each other. Calcium (Ca2+) coordination at the EC linker regions is key for tip-link elasticity and function. There are ∼116 sites in CDH23 affected by deafness-causing mutations, many of which alter conserved Ca2+-binding residues. Here we present crystal structures showing 18 CDH23 EC repeats, including the most and least conserved, a fragment carrying disease mutations, and EC repeats with non-canonical Ca2+-binding motif sequences and unusual secondary structure. Complementary experiments show deafness mutations' effects on stability and affinity for Ca2+. Additionally, a model of nine contiguous CDH23 EC repeats reveals helicity and potential parallel dimerization faces. Overall, our studies provide detailed structural insight into CDH23 function in mechanotransduction.

Tuning Inner-Ear Tip-Link Affinity Through Alternatively Spliced Variants of Protocadherin-15.

Human hearing relies upon the tip-to-tip interaction of two nonclassical cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23). Together, these proteins form a filament called the tip link that connects neighboring stereocilia of mechanosensitive hair cells. As sound waves enter the cochlea, the stereocilia deflect and tension is applied to the tip link, opening nearby transduction channels. Disruption of the tip link by loud sound or calcium chelators eliminates transduction currents and illustrates that tip-link integrity is critical for mechanosensing. Tip-link remodeling after disruption is a dynamic process, which can lead to the formation of atypical complexes that incorporate alternatively spliced variants of PCDH15. These variants are categorized into six groups (N1-N6) based upon differences in the first two extracellular cadherin (EC) repeats. Here, we characterized the two N-terminal EC repeats of all PCDH15 variants (pcdh15(N1) to pcdh15(N6)) and combined these variants to test complex formation. We solved the crystal structure of a new complex composed of CDH23 EC1-2 (cdh23) and pcdh15(N2) at 2.3 Å resolution and compared it to the canonical cdh23-pcdh15(N1) complex. While there were subtle structural differences, the binding affinity between cdh23 and pcdh15(N2) is ∼6 times weaker than cdh23 and pcdh15(N1) as determined by surface plasmon resonance analysis. Steered molecular dynamics simulations predict that the unbinding force of the cdh23-pcdh15(N2) complex can be lower than the canonical tip link. Our results demonstrate that alternative heterophilic tip-link structures form stable protein-protein interactions in vitro and suggest that homophilic PCDH15-PCDH15 tip links form through the interaction of additional EC repeats.

Neuron-specific knockdown of the Drosophila fat induces reduction of life span, deficient locomotive ability, shortening of motoneuron terminal branches and defects in axonal targeting.

Mutations in FAT4 gene, one of the human FAT family genes, have been identified in Van Maldergem syndrome (VMS) and Hennekam lymphangiectasia-lymphedema syndrome (HS). The FAT4 gene encodes a large protein with extracellular cadherin repeats, EGF-like domains and Laminin G-like domains. FAT4 plays a role in tumor suppression and planar cell polarity. Drosophila contains a human FAT4 homologue, fat. Drosophila fat has been mainly studied with Drosophila eye and wing systems. Here, we specially knocked down Drosophila fat in nerve system. Neuron-specific knockdown of fat shortened the life span and induced the defect in locomotive abilities of adult flies. In consistent with these phenotypes, defects in synapse structure at neuromuscular junction were observed in neuron-specific fat-knockdown flies. In addition, aberrations in axonal targeting of photoreceptor neuron in third-instar larvae were also observed, suggesting that fat involves in axonal targeting. Taken together, the results indicate that Drosophila fat plays an essential role in formation and/or maintenance of neuron. Both VMS and HS show mental retardation and neuronal defects. We therefore consider that these two rare human diseases could possibly be caused by the defect in FAT4 function in neuronal cells.

The unique prodomain of T-cadherin plays a key role in adiponectin binding with the essential extracellular cadherin repeats 1 and 2

Adiponectin, an adipocyte-derived circulating protein, accumulates in the heart, vascular endothelium, and skeletal muscles through an interaction with T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored cadherin. Recent studies have suggested that this interaction is essential for adiponectin-mediated cardiovascular protection. However, the precise protein-protein interaction between adiponectin and T-cad remains poorly characterized. Using ELISA-based and surface plasmon analyses, we report here that T-cad fused with IgG Fc as a fusion tag by replacing its glycosylphosphatidylinositol-anchor specifically bound both hexameric and larger multimeric adiponectin with a dissociation constant of ∼1.0 nm and without any contribution from other cellular or serum factors. The extracellular T-cad repeats 1 and 2 were critical for the observed adiponectin binding, which is required for classical cadherin-mediated cell-to-cell adhesion. Moreover, the 130-kDa prodomain-bearing T-cad, uniquely expressed on the cell surface among members of the cadherin family and predominantly increased by adiponectin, contributed significantly to adiponectin binding. Inhibition of prodomain-processing by a prohormone convertase inhibitor increased 130-kDa T-cad levels and also enhanced adiponectin binding to endothelial cells both by more preferential cell-surface localization and by higher adiponectin-binding affinity of 130-kDa T-cad relative to 100-kDa T-cad. The preferential cell-surface localization of 130-kDa T-cad relative to 100-kDa T-cad was also observed in normal mice aorta in vivo In conclusion, our study shows that a unique key feature of the T-cad prodomain is its involvement in binding of the T-cad repeats 1 and 2 to adiponectin and also demonstrates that adiponectin positively regulates T-cad abundance.

A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.