Site-directed mutagenesis and computational homology modeling were employed to identify critical residues within segments A and C that are essential for the binding of MEDI-565. The amino acids of segments A and C of the A2 domain that differed from the A3 domains were replaced with the corresponding A3 residues encoding several substitutions at a time: Figure 4A ). The binding of MEDI-565 was substantially decreased to the variants in which residue N 333 was mutated (KO_FTN and KO_NE), but bound well to the other mutants ( Figure 5A ). Furthermore, replacing only residue N 333 with either its counterpart residue Lys in the A3 domain (KO_N) or with Ala (N 333 to A) abolished MEDI-565 binding ( Figure 5C ). Mutating the residue F 326 or T 328 to Ala substantially decreased the binding of MEDI-565 (F 326 to A, T 328 to A), suggesting that they were also involved in the interaction with MEDI-565 but to a lesser extent than residue N 333 ( Figure 5C ). Taken together, our data demonstrated the importance of F 326 , T 328 , and N 333 . Finally, since E 338 is encoded in the KO_ELI variant which binds well to MEDI-565, we concluded that this residue is not energetically involved in MEDI-565 epitope. We had previously noted that grafting only segment A that includes the F 326 , T 328 , and N 333 residues of the A2 domain into the A3 domain (KI_A) did not result in MEDI-565 binding. This suggested that MEDI-565 binding epitope comprises additional critical residues and pointed towards it being nonlinear and conformational.