Atomic reconstruction of HAd3K/EC2-EC3 complex, comparison to CAR and CD46. a Side view of HAd3K/EC2-EC3 complex with the three HAd3K monomers colored in blue, gold and red and EC2-EC3 colored in orange. b Top view of the same complex. c Top view of HAd3K/(EC2-EC3)₂ with the second module in green. d Top view of HAd11 Knob structure with three CD46 SCR1&2 molecules in orange (PDB: 2O39). e Top view of HAd12 Knob with CAR D1 domain in orange (PDB: 1KAC)

Determination of residues from HAd3K and EC2-EC3 involved in the interaction. a Surface of EC2-EC3 interacting with HAd3K. Aminoacids making contact with HAd3K are colored in light or dark green according to their interaction with monomer A or B, respectively. For a better view only two monomers of HAd3K are represented in cartoon. b Surface of HAd3K involved in receptor binding. HAd3K aminoacids involved in binding are highlighted in dark purple (EC2 contacts) and light purple (EC3 contacts). c EC2-EC3 primary sequence with aminoacids making contact with the knob are colored using same color code than in a. d Primary sequence of HAd3K with β-strand depicted with arrows. Contact-forming residues of HAd3K are depicted with same colors as in b. Asterisks (*) denote residues for which single mutation reduce DSG2 binding by more than 80% as decribed in Wang et al.

A single D261A mutation in HAd3K is sufficient to abolish the receptor binding. a HAd3K incubation with an excess of EC2-EC3 produced in mammalian cell resulted in a complex eluted between 12 and 14 mL as shown by SEC and confirmed by SDS-PAGE. b Incubation of EC2-EC3 with the D261A mutated HAd3K resulted in a pic eluted latter (14–16 mL) corresponding to the two free components as shown by SDS-PAGE. Dotted rectangles on SDS-PAGE indicates the expected position of EC2-EC3 when in complex with the knob

Receptor cadherin domains twisting upon HAd3K binding. Superimposition of unliganded (in pink) and bound (in orange) EC2-EC3 to HAd3K (in grey) shows a 10° twist between EC2 and EC3