Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers
Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present cr...
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| creator | Jin, Xiangshu Walker, Melissa A. Felsövályi, Klára Vendome, Jeremie Bahna, Fabiana Mannepalli, Seetha Cosmanescu, Filip Ahlsen, Goran Honig, Barry Shapiro, Lawrence |
| description | Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from
Drosophila
N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1’) of the mature protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ pairs display binding of three Ca
2+
ions similar to that of vertebrate cadherins, domains EC2’ and EC3’ are joined in a “kinked” orientation by a previously uncharacterized Ca
2+
-free linker. Biophysical analysis demonstrates that a construct containing the predicted N-terminal nine EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins, whereas deleting the ninth EC domain ablates dimerization. These results suggest that, unlike their vertebrate counterparts, invertebrate cadherins may utilize multiple EC domains to form intercellular adhesive bonds. Sequence analysis reveals that similar Ca
2+
-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins.
Taken together, our findings provide a plausible answer to an intriguing question in cell adhesion: How do large cadherins containing numerous extracellular cadherin domains fit into intercellular spaces of comparable dimensions to those where the five-domain vertebrate classical cadherins mediate cell adhesion? Our results suggest that the unique Ca
2+
-free linkers observed in the structures of DN-cadherin ectodomain fragments and also found to be present in a large number of nonclassical cadherins may impart complex “folded” ectodomain architectures to these cadherins to facilitate their specific biological functions. These findings provide an important step forward in understanding cadherin-mediated cell adhesion in invertebrates, and also yield new insights into the structure and function of the numerous vertebrate cadherins with large ectodomains.
Based on the unique Ca
2+
-free interdomain linkage seen in the DN-cadherin crystal structures, we performed bioinformatic analyses of the entire cadherin superfamily. Within the set of 23,340 cadherin domains identified in 3,673 proteins, we found 2,504 linkers with different combinations of missing Ca
2+ |
| doi_str_mv | 10.1073/pnas.1117538108 |
| format | Article |
| fullrecord | <record><control><sourceid>crossref</sourceid><recordid>TN_cdi_crossref_primary_10_1073_pnas_1117538108</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>10_1073_pnas_1117538108</sourcerecordid><originalsourceid>FETCH-LOGICAL-c1278-bb25838d7000e8a33b3b114c2109ab4ac4ed0b2c18935ce93e1a2967eda531803</originalsourceid><addsrcrecordid>eNpFkL1PwzAUxC0EEqUws3pHbt-zk8YZUfiUKlhgjl6cF2pIk8pOQZX440mhEtPdcqe7nxCXCDOEzMw3HcUZImapsQj2SEwQclSLJIdjMQHQmbKJTk7FWYzvAJCnFibiuwi7OFAr4xC2btgGjrJv5E3oY79Z-Zbkk3JUrzj4TrIb-rpf02gDv_m-i6N-8pgm-eVrbndyG7mWrqX4W1OQ1FdSNYFZ-m7gcEi3vvvgEM_FSUNt5IuDTsXr3e1L8aCWz_ePxfVSOdSZVVWlU2tsnY2r2ZIxlakQE6fHg1Ql5BKuodIObW5Sx7lhJJ0vMq4pNWjBTMX8r9eNt2LgptwEv6awKxHKPbxyD6_8h2d-ADoHZFM</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers</title><source>JSTOR Archive Collection A-Z Listing</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><source>Free Full-Text Journals in Chemistry</source><creator>Jin, Xiangshu ; Walker, Melissa A. ; Felsövályi, Klára ; Vendome, Jeremie ; Bahna, Fabiana ; Mannepalli, Seetha ; Cosmanescu, Filip ; Ahlsen, Goran ; Honig, Barry ; Shapiro, Lawrence</creator><creatorcontrib>Jin, Xiangshu ; Walker, Melissa A. ; Felsövályi, Klára ; Vendome, Jeremie ; Bahna, Fabiana ; Mannepalli, Seetha ; Cosmanescu, Filip ; Ahlsen, Goran ; Honig, Barry ; Shapiro, Lawrence</creatorcontrib><description>Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from
Drosophila
N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1’) of the mature protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ pairs display binding of three Ca
2+
ions similar to that of vertebrate cadherins, domains EC2’ and EC3’ are joined in a “kinked” orientation by a previously uncharacterized Ca
2+
-free linker. Biophysical analysis demonstrates that a construct containing the predicted N-terminal nine EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins, whereas deleting the ninth EC domain ablates dimerization. These results suggest that, unlike their vertebrate counterparts, invertebrate cadherins may utilize multiple EC domains to form intercellular adhesive bonds. Sequence analysis reveals that similar Ca
2+
-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins.
Taken together, our findings provide a plausible answer to an intriguing question in cell adhesion: How do large cadherins containing numerous extracellular cadherin domains fit into intercellular spaces of comparable dimensions to those where the five-domain vertebrate classical cadherins mediate cell adhesion? Our results suggest that the unique Ca
2+
-free linkers observed in the structures of DN-cadherin ectodomain fragments and also found to be present in a large number of nonclassical cadherins may impart complex “folded” ectodomain architectures to these cadherins to facilitate their specific biological functions. These findings provide an important step forward in understanding cadherin-mediated cell adhesion in invertebrates, and also yield new insights into the structure and function of the numerous vertebrate cadherins with large ectodomains.
Based on the unique Ca
2+
-free interdomain linkage seen in the DN-cadherin crystal structures, we performed bioinformatic analyses of the entire cadherin superfamily. Within the set of 23,340 cadherin domains identified in 3,673 proteins, we found 2,504 linkers with different combinations of missing Ca
2+
-binding residues distributed in 936 proteins, most of which are nonclassical cadherins containing a large number of extracellular cadherin domains, including DN- and DE-related cadherins, Fat, Dachsous, Flamingo/CELSR, and choanoflagellate cadherins. On the whole superfamily scale, the larger the cadherins, the more Ca
2+
-free linkers they contain, and within a given subfamily, the distribution and pattern of the Ca
2+
-free linker types are generally conserved or similar across species.
In agreement with the crystallographic observations, additional analysis showed that DN-cadherin ectodomain fragments containing domains EC1′–EC4′ are monomers in solution. A larger construct that includes nine N-terminal extracellular cadherin domains forms homodimers (complexes composed of a pair of identical proteins) with an affinity similar to that of vertebrate classical cadherins, whereas deleting the ninth extracellular cadherin domain abolishes dimerization, suggesting that the DN-cadherin adhesive interface involves at least a portion of the EC1′–EC9′ region. Collectively, our data suggest that DN-cadherin and related cadherins may function in cell adhesion through binding interfaces involving several extracellular cadherin domains within the EC1′–EC9′ region via a mechanism distinct from that of vertebrate classical cadherins, which bridge the intercellular space by an elongated arrangement of extracellular cadherin repeats with binding interfaces confined to their distal N-terminal EC1 domains (
5
).
To gain insight into the structural basis for how DN-cadherin functions in cell adhesion, we determined the structures of DN-cadherin ectodomain regions corresponding to the four predicted N-terminal extracellular cadherin domains (denoted EC1′–EC4′) in the mature protein. The overall architecture of the DN-cadherin EC1′–EC4′ fragment differs significantly from vertebrate cadherin ectodomains, which adopt an elongated curved structure (
4
,
5
). The DN-cadherin EC1′–EC4′ fragment adopts a V-shaped structure (
Fig. P1
) with a prominent “kink” between the EC2′ and EC3′ domains, which are connected by a short loop comprising two consecutive Glycine residues instead of a typical linker with Ca
2+
ions bound to it. The EC2′–EC3′ interdomain interface facilitated by this unique Ca
2+
-free linker is consistent in all three structures determined for the DN-cadherin EC1′–EC4′ or EC1′–EC3′ fragment in three distinct crystal forms, suggesting that it may be a stable structural feature required for the proper folding and adhesive functions of DN-cadherin. The linker region between the DN-cadherin EC1′ and EC2′ domains and that between the EC3′ and EC4′ domain pairs each contains three calcium ions bound in a way similar to that seen in vertebrate classical cadherins. However, the DN-cadherin EC1′ domain does not engage in strand-swap, the primary feature of vertebrate classical cadherins’ adhesive dimerization, in which the N-terminal β-strands of paired EC1 domains replace one another. Instead, the N-terminal strand of DN-cadherin EC1′ domain is entirely integrated into the main body of the EC1′ domain, consistent with the absence of sequence features required for strand-swap dimerization in vertebrate classical cadherins.
Fig. P1.
The DN-cadherin EC1′–EC4′ ectodomain region adopts a V-shaped structure imparted by a unique Ca
2+
-free linker (red arrow) between domains EC2′ and EC3′. The linker region between the EC1′ and EC2′ domains and that between the EC3′ and EC4′ domains contain three calcium ions (green spheres) similar to vertebrate classical cadherins.
Cell adhesion is a distinguishing feature of metazoan (i.e., animal) species and is essential to the development and maintenance of solid tissues. Calcium-dependent cell adhesion is mediated primarily by cadherins (
1
), a family of cell surface proteins. Cadherins are characterized by the presence of extracellular cadherin domains, modules of about 110 amino acids that form a β-sandwich fold with a Greek key topology. The best characterized cadherins are vertebrate classical cadherins, a family of molecules that share similar domain structures. These structures each consist of an ectodomain (i.e., a protein domain extending into the space outside the cell) with five tandem extracellular cadherin domains that are rigidified by the binding of three Ca
2+
ions between each successive pair of extracellular cadherin domains; a region spanning the cell membrane; and a short, conserved tail in the cytoplasm, the interior of the cell (
1
). Invertebrate cadherins are highly divergent from vertebrate counterparts, yet play similar roles in cell adhesion. For example, the
Drosophila
N- and E- (DN- and DE-) cadherins serve functions in cell adhesion and tissue patterning, analogous to their vertebrate counterparts (
2
,
3
). However, the ectodomains of DN- and DE-cadherins differ markedly from those of their vertebrate counterparts both in size and particular features. Thus, the mechanistic understanding of ectodomain-mediated adhesion that has been gained for vertebrate classical cadherins is unlikely to apply to DN-cadherin and other related cadherins. How these invertebrate cadherins with large ectodomains perform their adhesive function at intercellular junctions is unclear, because the distances between cells in both vertebrate and invertebrate tissues are similar and can be spanned by only five extracellular cadherin domains per molecule in vertebrate species.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1117538108</identifier><language>eng</language><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2012-01, Vol.109 (3)</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c1278-bb25838d7000e8a33b3b114c2109ab4ac4ed0b2c18935ce93e1a2967eda531803</citedby><cites>FETCH-LOGICAL-c1278-bb25838d7000e8a33b3b114c2109ab4ac4ed0b2c18935ce93e1a2967eda531803</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids></links><search><creatorcontrib>Jin, Xiangshu</creatorcontrib><creatorcontrib>Walker, Melissa A.</creatorcontrib><creatorcontrib>Felsövályi, Klára</creatorcontrib><creatorcontrib>Vendome, Jeremie</creatorcontrib><creatorcontrib>Bahna, Fabiana</creatorcontrib><creatorcontrib>Mannepalli, Seetha</creatorcontrib><creatorcontrib>Cosmanescu, Filip</creatorcontrib><creatorcontrib>Ahlsen, Goran</creatorcontrib><creatorcontrib>Honig, Barry</creatorcontrib><creatorcontrib>Shapiro, Lawrence</creatorcontrib><title>Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers</title><title>Proceedings of the National Academy of Sciences - PNAS</title><description>Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from
Drosophila
N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1’) of the mature protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ pairs display binding of three Ca
2+
ions similar to that of vertebrate cadherins, domains EC2’ and EC3’ are joined in a “kinked” orientation by a previously uncharacterized Ca
2+
-free linker. Biophysical analysis demonstrates that a construct containing the predicted N-terminal nine EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins, whereas deleting the ninth EC domain ablates dimerization. These results suggest that, unlike their vertebrate counterparts, invertebrate cadherins may utilize multiple EC domains to form intercellular adhesive bonds. Sequence analysis reveals that similar Ca
2+
-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins.
Taken together, our findings provide a plausible answer to an intriguing question in cell adhesion: How do large cadherins containing numerous extracellular cadherin domains fit into intercellular spaces of comparable dimensions to those where the five-domain vertebrate classical cadherins mediate cell adhesion? Our results suggest that the unique Ca
2+
-free linkers observed in the structures of DN-cadherin ectodomain fragments and also found to be present in a large number of nonclassical cadherins may impart complex “folded” ectodomain architectures to these cadherins to facilitate their specific biological functions. These findings provide an important step forward in understanding cadherin-mediated cell adhesion in invertebrates, and also yield new insights into the structure and function of the numerous vertebrate cadherins with large ectodomains.
Based on the unique Ca
2+
-free interdomain linkage seen in the DN-cadherin crystal structures, we performed bioinformatic analyses of the entire cadherin superfamily. Within the set of 23,340 cadherin domains identified in 3,673 proteins, we found 2,504 linkers with different combinations of missing Ca
2+
-binding residues distributed in 936 proteins, most of which are nonclassical cadherins containing a large number of extracellular cadherin domains, including DN- and DE-related cadherins, Fat, Dachsous, Flamingo/CELSR, and choanoflagellate cadherins. On the whole superfamily scale, the larger the cadherins, the more Ca
2+
-free linkers they contain, and within a given subfamily, the distribution and pattern of the Ca
2+
-free linker types are generally conserved or similar across species.
In agreement with the crystallographic observations, additional analysis showed that DN-cadherin ectodomain fragments containing domains EC1′–EC4′ are monomers in solution. A larger construct that includes nine N-terminal extracellular cadherin domains forms homodimers (complexes composed of a pair of identical proteins) with an affinity similar to that of vertebrate classical cadherins, whereas deleting the ninth extracellular cadherin domain abolishes dimerization, suggesting that the DN-cadherin adhesive interface involves at least a portion of the EC1′–EC9′ region. Collectively, our data suggest that DN-cadherin and related cadherins may function in cell adhesion through binding interfaces involving several extracellular cadherin domains within the EC1′–EC9′ region via a mechanism distinct from that of vertebrate classical cadherins, which bridge the intercellular space by an elongated arrangement of extracellular cadherin repeats with binding interfaces confined to their distal N-terminal EC1 domains (
5
).
To gain insight into the structural basis for how DN-cadherin functions in cell adhesion, we determined the structures of DN-cadherin ectodomain regions corresponding to the four predicted N-terminal extracellular cadherin domains (denoted EC1′–EC4′) in the mature protein. The overall architecture of the DN-cadherin EC1′–EC4′ fragment differs significantly from vertebrate cadherin ectodomains, which adopt an elongated curved structure (
4
,
5
). The DN-cadherin EC1′–EC4′ fragment adopts a V-shaped structure (
Fig. P1
) with a prominent “kink” between the EC2′ and EC3′ domains, which are connected by a short loop comprising two consecutive Glycine residues instead of a typical linker with Ca
2+
ions bound to it. The EC2′–EC3′ interdomain interface facilitated by this unique Ca
2+
-free linker is consistent in all three structures determined for the DN-cadherin EC1′–EC4′ or EC1′–EC3′ fragment in three distinct crystal forms, suggesting that it may be a stable structural feature required for the proper folding and adhesive functions of DN-cadherin. The linker region between the DN-cadherin EC1′ and EC2′ domains and that between the EC3′ and EC4′ domain pairs each contains three calcium ions bound in a way similar to that seen in vertebrate classical cadherins. However, the DN-cadherin EC1′ domain does not engage in strand-swap, the primary feature of vertebrate classical cadherins’ adhesive dimerization, in which the N-terminal β-strands of paired EC1 domains replace one another. Instead, the N-terminal strand of DN-cadherin EC1′ domain is entirely integrated into the main body of the EC1′ domain, consistent with the absence of sequence features required for strand-swap dimerization in vertebrate classical cadherins.
Fig. P1.
The DN-cadherin EC1′–EC4′ ectodomain region adopts a V-shaped structure imparted by a unique Ca
2+
-free linker (red arrow) between domains EC2′ and EC3′. The linker region between the EC1′ and EC2′ domains and that between the EC3′ and EC4′ domains contain three calcium ions (green spheres) similar to vertebrate classical cadherins.
Cell adhesion is a distinguishing feature of metazoan (i.e., animal) species and is essential to the development and maintenance of solid tissues. Calcium-dependent cell adhesion is mediated primarily by cadherins (
1
), a family of cell surface proteins. Cadherins are characterized by the presence of extracellular cadherin domains, modules of about 110 amino acids that form a β-sandwich fold with a Greek key topology. The best characterized cadherins are vertebrate classical cadherins, a family of molecules that share similar domain structures. These structures each consist of an ectodomain (i.e., a protein domain extending into the space outside the cell) with five tandem extracellular cadherin domains that are rigidified by the binding of three Ca
2+
ions between each successive pair of extracellular cadherin domains; a region spanning the cell membrane; and a short, conserved tail in the cytoplasm, the interior of the cell (
1
). Invertebrate cadherins are highly divergent from vertebrate counterparts, yet play similar roles in cell adhesion. For example, the
Drosophila
N- and E- (DN- and DE-) cadherins serve functions in cell adhesion and tissue patterning, analogous to their vertebrate counterparts (
2
,
3
). However, the ectodomains of DN- and DE-cadherins differ markedly from those of their vertebrate counterparts both in size and particular features. Thus, the mechanistic understanding of ectodomain-mediated adhesion that has been gained for vertebrate classical cadherins is unlikely to apply to DN-cadherin and other related cadherins. How these invertebrate cadherins with large ectodomains perform their adhesive function at intercellular junctions is unclear, because the distances between cells in both vertebrate and invertebrate tissues are similar and can be spanned by only five extracellular cadherin domains per molecule in vertebrate species.</description><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><recordid>eNpFkL1PwzAUxC0EEqUws3pHbt-zk8YZUfiUKlhgjl6cF2pIk8pOQZX440mhEtPdcqe7nxCXCDOEzMw3HcUZImapsQj2SEwQclSLJIdjMQHQmbKJTk7FWYzvAJCnFibiuwi7OFAr4xC2btgGjrJv5E3oY79Z-Zbkk3JUrzj4TrIb-rpf02gDv_m-i6N-8pgm-eVrbndyG7mWrqX4W1OQ1FdSNYFZ-m7gcEi3vvvgEM_FSUNt5IuDTsXr3e1L8aCWz_ePxfVSOdSZVVWlU2tsnY2r2ZIxlakQE6fHg1Ql5BKuodIObW5Sx7lhJJ0vMq4pNWjBTMX8r9eNt2LgptwEv6awKxHKPbxyD6_8h2d-ADoHZFM</recordid><startdate>20120117</startdate><enddate>20120117</enddate><creator>Jin, Xiangshu</creator><creator>Walker, Melissa A.</creator><creator>Felsövályi, Klára</creator><creator>Vendome, Jeremie</creator><creator>Bahna, Fabiana</creator><creator>Mannepalli, Seetha</creator><creator>Cosmanescu, Filip</creator><creator>Ahlsen, Goran</creator><creator>Honig, Barry</creator><creator>Shapiro, Lawrence</creator><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>20120117</creationdate><title>Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers</title><author>Jin, Xiangshu ; Walker, Melissa A. ; Felsövályi, Klára ; Vendome, Jeremie ; Bahna, Fabiana ; Mannepalli, Seetha ; Cosmanescu, Filip ; Ahlsen, Goran ; Honig, Barry ; Shapiro, Lawrence</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c1278-bb25838d7000e8a33b3b114c2109ab4ac4ed0b2c18935ce93e1a2967eda531803</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jin, Xiangshu</creatorcontrib><creatorcontrib>Walker, Melissa A.</creatorcontrib><creatorcontrib>Felsövályi, Klára</creatorcontrib><creatorcontrib>Vendome, Jeremie</creatorcontrib><creatorcontrib>Bahna, Fabiana</creatorcontrib><creatorcontrib>Mannepalli, Seetha</creatorcontrib><creatorcontrib>Cosmanescu, Filip</creatorcontrib><creatorcontrib>Ahlsen, Goran</creatorcontrib><creatorcontrib>Honig, Barry</creatorcontrib><creatorcontrib>Shapiro, Lawrence</creatorcontrib><collection>CrossRef</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jin, Xiangshu</au><au>Walker, Melissa A.</au><au>Felsövályi, Klára</au><au>Vendome, Jeremie</au><au>Bahna, Fabiana</au><au>Mannepalli, Seetha</au><au>Cosmanescu, Filip</au><au>Ahlsen, Goran</au><au>Honig, Barry</au><au>Shapiro, Lawrence</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><date>2012-01-17</date><risdate>2012</risdate><volume>109</volume><issue>3</issue><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from
Drosophila
N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1’) of the mature protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ pairs display binding of three Ca
2+
ions similar to that of vertebrate cadherins, domains EC2’ and EC3’ are joined in a “kinked” orientation by a previously uncharacterized Ca
2+
-free linker. Biophysical analysis demonstrates that a construct containing the predicted N-terminal nine EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins, whereas deleting the ninth EC domain ablates dimerization. These results suggest that, unlike their vertebrate counterparts, invertebrate cadherins may utilize multiple EC domains to form intercellular adhesive bonds. Sequence analysis reveals that similar Ca
2+
-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins.
Taken together, our findings provide a plausible answer to an intriguing question in cell adhesion: How do large cadherins containing numerous extracellular cadherin domains fit into intercellular spaces of comparable dimensions to those where the five-domain vertebrate classical cadherins mediate cell adhesion? Our results suggest that the unique Ca
2+
-free linkers observed in the structures of DN-cadherin ectodomain fragments and also found to be present in a large number of nonclassical cadherins may impart complex “folded” ectodomain architectures to these cadherins to facilitate their specific biological functions. These findings provide an important step forward in understanding cadherin-mediated cell adhesion in invertebrates, and also yield new insights into the structure and function of the numerous vertebrate cadherins with large ectodomains.
Based on the unique Ca
2+
-free interdomain linkage seen in the DN-cadherin crystal structures, we performed bioinformatic analyses of the entire cadherin superfamily. Within the set of 23,340 cadherin domains identified in 3,673 proteins, we found 2,504 linkers with different combinations of missing Ca
2+
-binding residues distributed in 936 proteins, most of which are nonclassical cadherins containing a large number of extracellular cadherin domains, including DN- and DE-related cadherins, Fat, Dachsous, Flamingo/CELSR, and choanoflagellate cadherins. On the whole superfamily scale, the larger the cadherins, the more Ca
2+
-free linkers they contain, and within a given subfamily, the distribution and pattern of the Ca
2+
-free linker types are generally conserved or similar across species.
In agreement with the crystallographic observations, additional analysis showed that DN-cadherin ectodomain fragments containing domains EC1′–EC4′ are monomers in solution. A larger construct that includes nine N-terminal extracellular cadherin domains forms homodimers (complexes composed of a pair of identical proteins) with an affinity similar to that of vertebrate classical cadherins, whereas deleting the ninth extracellular cadherin domain abolishes dimerization, suggesting that the DN-cadherin adhesive interface involves at least a portion of the EC1′–EC9′ region. Collectively, our data suggest that DN-cadherin and related cadherins may function in cell adhesion through binding interfaces involving several extracellular cadherin domains within the EC1′–EC9′ region via a mechanism distinct from that of vertebrate classical cadherins, which bridge the intercellular space by an elongated arrangement of extracellular cadherin repeats with binding interfaces confined to their distal N-terminal EC1 domains (
5
).
To gain insight into the structural basis for how DN-cadherin functions in cell adhesion, we determined the structures of DN-cadherin ectodomain regions corresponding to the four predicted N-terminal extracellular cadherin domains (denoted EC1′–EC4′) in the mature protein. The overall architecture of the DN-cadherin EC1′–EC4′ fragment differs significantly from vertebrate cadherin ectodomains, which adopt an elongated curved structure (
4
,
5
). The DN-cadherin EC1′–EC4′ fragment adopts a V-shaped structure (
Fig. P1
) with a prominent “kink” between the EC2′ and EC3′ domains, which are connected by a short loop comprising two consecutive Glycine residues instead of a typical linker with Ca
2+
ions bound to it. The EC2′–EC3′ interdomain interface facilitated by this unique Ca
2+
-free linker is consistent in all three structures determined for the DN-cadherin EC1′–EC4′ or EC1′–EC3′ fragment in three distinct crystal forms, suggesting that it may be a stable structural feature required for the proper folding and adhesive functions of DN-cadherin. The linker region between the DN-cadherin EC1′ and EC2′ domains and that between the EC3′ and EC4′ domain pairs each contains three calcium ions bound in a way similar to that seen in vertebrate classical cadherins. However, the DN-cadherin EC1′ domain does not engage in strand-swap, the primary feature of vertebrate classical cadherins’ adhesive dimerization, in which the N-terminal β-strands of paired EC1 domains replace one another. Instead, the N-terminal strand of DN-cadherin EC1′ domain is entirely integrated into the main body of the EC1′ domain, consistent with the absence of sequence features required for strand-swap dimerization in vertebrate classical cadherins.
Fig. P1.
The DN-cadherin EC1′–EC4′ ectodomain region adopts a V-shaped structure imparted by a unique Ca
2+
-free linker (red arrow) between domains EC2′ and EC3′. The linker region between the EC1′ and EC2′ domains and that between the EC3′ and EC4′ domains contain three calcium ions (green spheres) similar to vertebrate classical cadherins.
Cell adhesion is a distinguishing feature of metazoan (i.e., animal) species and is essential to the development and maintenance of solid tissues. Calcium-dependent cell adhesion is mediated primarily by cadherins (
1
), a family of cell surface proteins. Cadherins are characterized by the presence of extracellular cadherin domains, modules of about 110 amino acids that form a β-sandwich fold with a Greek key topology. The best characterized cadherins are vertebrate classical cadherins, a family of molecules that share similar domain structures. These structures each consist of an ectodomain (i.e., a protein domain extending into the space outside the cell) with five tandem extracellular cadherin domains that are rigidified by the binding of three Ca
2+
ions between each successive pair of extracellular cadherin domains; a region spanning the cell membrane; and a short, conserved tail in the cytoplasm, the interior of the cell (
1
). Invertebrate cadherins are highly divergent from vertebrate counterparts, yet play similar roles in cell adhesion. For example, the
Drosophila
N- and E- (DN- and DE-) cadherins serve functions in cell adhesion and tissue patterning, analogous to their vertebrate counterparts (
2
,
3
). However, the ectodomains of DN- and DE-cadherins differ markedly from those of their vertebrate counterparts both in size and particular features. Thus, the mechanistic understanding of ectodomain-mediated adhesion that has been gained for vertebrate classical cadherins is unlikely to apply to DN-cadherin and other related cadherins. How these invertebrate cadherins with large ectodomains perform their adhesive function at intercellular junctions is unclear, because the distances between cells in both vertebrate and invertebrate tissues are similar and can be spanned by only five extracellular cadherin domains per molecule in vertebrate species.</abstract><doi>10.1073/pnas.1117538108</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0027-8424 |
| ispartof | Proceedings of the National Academy of Sciences - PNAS, 2012-01, Vol.109 (3) |
| issn | 0027-8424 1091-6490 |
| language | eng |
| recordid | cdi_crossref_primary_10_1073_pnas_1117538108 |
| source | JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| title | Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-27T03%3A22%3A55IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-crossref&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Crystal%20structures%20of%20Drosophila%20N-cadherin%20ectodomain%20regions%20reveal%20a%20widely%20used%20class%20of%20Ca%202+%20-free%20interdomain%20linkers&rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20-%20PNAS&rft.au=Jin,%20Xiangshu&rft.date=2012-01-17&rft.volume=109&rft.issue=3&rft.issn=0027-8424&rft.eissn=1091-6490&rft_id=info:doi/10.1073/pnas.1117538108&rft_dat=%3Ccrossref%3E10_1073_pnas_1117538108%3C/crossref%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/&rfr_iscdi=true |