The calcium-binding epidermal growth factor-like (cbEGF) domain is a widely occurring module in proteins of diverse function. Amino acid substitutions that disrupt its structure or calcium affinity have been associated with various disorders. The extracellular portion of CRB1, the human homologue of Drosophila Crumbs, exhibits a modular domain organization that includes EGF and cbEGF domains. The N1317H substitution in the 19th cbEGF domain of CRB1 is associated with the serious visual disorder Leber congenital amaurosis. We have investigated the structure and Ca(2+) binding of recombinant wild-type and N1317H CRB1 fragments (EGF18-cbEGF19) using NMR and find that Ca(2+) binding is altered, resulting in disruption of long range interactions between adjacent EGF domains in CRB1. From these observations, we propose that this substitution affects the structural integrity of CRB1 in the inter-photoreceptor matrix of the retina, where it is expressed. Furthermore, we identify disease-causing substitutions in other cbEGF-containing proteins that are likely to result in similar disruption of interdomain packing, supporting the hypothesis that the tandem cbEGF domain linkages are critical for the structure and function of proteins containing cbEGF domains.