Mutation-induced dimerization of transforming growth factor-β–induced protein may drive protein aggregation in granular corneal dystrophy

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β–induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography–small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD

Structural and functional implications of human transforming growth factor b-Induced protein, TGFBIp, in corneal dystrophies

A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor β-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded β sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-β cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4This study was funded in part by grants from Spanish (BFU2015-64487-R and MDM-2014-0435) and Catalan (2014SGR9) public agencies, as well as from the Danish Council for Independent Research, Medical Sciences (DFF-4004- 00471), the Lundbeck Foundation (R164-2013-15912), the Velux Foundation, and Fight for Sight, Denmark. T.G. acknowledges a ‘‘Juan de la Cierva’’ research contract (JCI-2012-13573) from the Spanish Ministry for Economy and Competitiveness. The Structural Biology Unit of IBMB is a ‘‘Marı´a de Maeztu’’ Unit of Excellence of the Spanish Ministry of Economy, Industry and Competitiveness. Funding for data collection was provided in part by ESRFPeer reviewe