Structure-Function of Type IV Collagen NC1 Domains

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DESCRIPTION (provided by applicant): The structure of glomerular basement membrane (GBM) is altered in three notable diseases that affect the kidneys: Goodpasture syndrome (anti-GBM nephritis), Alport syndrome (hereditary nephritis), and diabetes mellitus. At least two of them, Goodpasture and Alport syndromes, have been linked to type IV collagen, a major constituent of GBM. Type IV collagen is a family of six alpha chains, alpha1-alpha6, found in various basement membranes (BMs). Three alpha chains form a collagen triple helical protomer and each chain contains a noncollagenous (NC1) globular domain at the C-terminus. The chain stoichiometry of the protomer varies with the origin of the tissue, (alpha1)2.alpha2 being the ubiquitous form and alpha3.alpha4.alpha5 being specific to glomeruli and alveoli. Two triple helical protomers associate at the NC1 region to form tail-to-tail dimer. The NC1 domains are presumed to contain the structural determinants for both triple helix formation and hexamer assembly. The alpha3 NC1 domain harbors the epitope for autoantibodies in patients with Goodpasture syndrome. Mutations in the genes encoding any of the three alpha chains result in the loss of alpha3.alpha4.alpha5 network in the GBM, which is the cause of Alport syndrome. Recombinant alpha2, alpha3, and alpha6 NC1 domains show anti-angiogenic and anti-tumor properties. In this study, we propose the following specific aims to determine the structure-function relationships of NC1 domains: Aim 1: To determine the first crystal structure of a NC1 hexamer, ((alpha1)2.alpha2)2 Aim 2: To crystallize and determine the structure of GBM NC1 hexamer, (alpha3.alpha4.alpha5)2 Aim 3: To determine the structure of the Goodpasture antigen, alpha3 NC1 monomer Aim 4: To solve the structures of alpha2 and alpha6 monomers, the angiogenic inhibitors. The accomplishment of these aims requires the application of macromolecular crystallography, computational biology, protein chemistry and molecular biology.