Fig. 5: Biophysical Characterization of COP-1 Binding.
From: Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment
a COP-1 binds to claudins in the absence of enterotoxins. COP-1 binding is maintained best with hsCLDN-4 but weaker with other subtypes. b COP-1 binding to claudin/cCpE complexes shows improved binding capacity compared to claudin alone for most subtypes assayed; hsCLDN-3 (light blue trace) is an exception. Note that the hierarchy of best binding subtypes is similar to claudins in absence of cCpE. c Sequence alignment of homologous claudins in their COP-1 binding epitopes. Because epitope 3 was 100% conserved, regions in epitopes 1 and 2 were mutated in hsCLDN-4 and -9 (pink dashed boxes) to validate structures. d COP-1 binding to mutant claudin/cCpE complexes shows that all hsCLDN-4 mutants lose binding capacity for COP-1, while for hsCLDN-9, mutations to epitope 2 (pink trace) lose binding while a Leu29Met point mutant in epitope 1 (dark pink trace) gains binding capacity to near wild type hsCLDN-4 (blue trace) levels. e Wild type hsCLDN-4 was reconstituted in synthetic amphipol polymers or lipid nanodiscs to test COP-1 binding in non-detergent systems. Results show that COP-1 binds to hsCLDN-4/cCpE complexes in each membrane mimetic but the kinetics of binding are altered.
