Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.