A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes

Abstract

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. II. Crystal structure of the α345 hexamer

“…2 and 3 ). The atomic structure is homologous to the crystal structure of the α121 NC1 domain ( 5 ) ( Fig. 3 ), which is also homologous to all reported crystal structures of tissue extracted from the human and bovine α121 NC1 domain ( 9 , 10 , 11 ).…”

“…After decades of attempts to isolate and crystallize the α345 hexamer, we developed a recombinant single-chain NC1 trimer technology ( 5 ) and used it to define the arrangement of chains and solve the crystal structure of the recombinant α345 hexamer.…”

“…We tested these possibilities experimentally using the single-chain NC1 trimer technology ( 5 ) developed and verified for the α121 hexamer. This technology allows to define composition and orientation of the chains in the trimer ( Fig.…”

Sequence-dependent mechanics of collagen reflect its structural and functional organization

“…Although imaging in liquid conditions is possible with AFM, here, we deposited the collagen from the specified solution conditions and dried prior to imaging. This follows previous work that used AFM to image and quantify the flexibility of collagen types I, II and III (34,35), to observe interactions between the NC1 domains of collagen IV (36), and to observe binding sites of laminin on collagen IV (37).…”

“…2A, along with an image of collagen I for comparison. The acidic conditions preclude lateral assembly of both fibril-forming and collagen IV molecules (27,34), while the presence of chloride ions induces oligomerization of collagen IV protomers, forming networks (13,36). Both proteins possess long chains, representing the collagenous domains.…”