title: Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity.
abstract: This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: A
authors.initials: A
authors.lastname: Prakasam
authors.forename: Y-H
authors.initials: YH
authors.lastname: Chien
authors.forename: V
authors.initials: V
authors.lastname: Maruthamuthu
authors.forename: D E
authors.initials: DE
authors.lastname: Leckband
mesh_terms: D000818:Animals; D001665:Binding Sites; D015820:Cadherins / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D002448:Cell Adhesion; D004285:Dogs; D008051:Lipid Bilayers / Q000737:chemistry; D008863:Microspheres; D009154:Mutation; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D015820:Cadherins; D008051:Lipid Bilayers; D013205:Staphylococcal Protein A; D014364:Tryptophan; D002118:Calcium