The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch-5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch-5b2, where dominant mutations leading to DFNA9 deafness disorder have been identi®ed. The fold is novel. Four of the ®ve known DFNA9 mutations are shown to involve at least partially solvent-exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent-accessible residues might be attributed to interference with the folding pathway of this disul®de-containing domain. Keywords: deafness disorder/DFNA9/LCCL domain/ NMR structure
IntroductionThe LCCL domain was identi®ed as an autonomous folding domain ®rst by amino acid sequence comparisons and subsequently by circular dichroism (CD) of an overexpressed construct (Trexler et al., 2000). It is a module named after its presence in Limulus factor C, cochlear protein Coch-5b2, and late gestation lung protein Lgl1, but it has also been found in several other proteins ( Figure 1). The LCCL domain plays a central role in the autosomal dominant human deafness disorder DFNA9, a non-syndromic sensorineural deafness associated with vestibular dysfunction, resulting in progressive hearing loss. Molecular analysis of cases of DFNA9 have identi®ed several unrelated kindreds with ®ve different mutations in the human COCH gene (Robertson et al., 1997(Robertson et al., , 1998de Kok et al., 1999;Fransen et al., 1999;Kamarinos et al., 2001). All these mutations are in the LCCL domain of Coch-5b2.The function of the LCCL domain seems to be either structural or antimicrobial. The domain is found in extracellular proteins in conjunction with other modular domains, including EGF-like, complement B-type, serine protease, von Willebrand type A, CAP, CUB, discoidinlectin and C-type lectin modules (Trexler et al., 2000). In Coch-5b2, as in the predicted protein from the related gene AAF19243_1 (Figure 1), the LCCL domain is joined to two domains with extensive homology to the collagenbinding type A domains of von Willebrand factor (Robertson et al., 1997(Robertson et al., , 1998. von Willebrand type A domains are present in a variety of extracellular matrix components, e.g. cartilage matrix protein and collagens type VI, VII, XII and XIV, suggesting that Coch-5b2 plays a structural role in the architecture of the cochlea by binding to components of the extracellular matrix of this sensory organ (Robertson et al., 1997(Robertson et al., , 1998. von Willebrand type A domains are, however, also found in proteins involved in defense mechanisms. For example, the von Willebrand factor itself is involved in hemostasis, th...