Table 1.
Data collection and refinement statistics.
Fig 1.
Comparison of panitumumab and cetuximab binding.
(A) Surface plasmon resonance of EGFR binding to panitumumab and cetuximab. Increasing concentrations of EGFR construct (1.23, 3.7, 11.1, 33.3 and 100 nM) were injected over immobilized antibody. In black and blue are the raw binding traces of the two different EGFR constructs and in red is data fit to a 1:1 binding model. (B) No binding was observed for cetuximab and EGFRD3 ND2 (S468R), blue line. (C) Overlay of panitumumab and EGFRD3 ND2 is shown in orange, panitumumab and EGFRD3 ND2(S468R) in magenta and cetuximab and EGFR domain III in cyan. (PDB: 1YY9). Only EGFR domain III (not domains I, II, and IV) is shown in the cetuximab complex for clarity. Panitumumab and cetuximab recognize the same epitope.
Fig 2.
Paratope surfaces and CDR interactions.
(A) Electrostatic surface of the CDR region of panitumumab. Large negatively charged cavity in center of CDR regions is clearly visible. (B) Electrostatic surface of CDR region for cetuximab reveals that Y104 fills the central cavity. (C) Light chain CDR interactions with the final β-strand of EGFR domain III. EGFR in tan with L1, L2 and L3 colored gray, purple, and yellow, respectively. (D) Heavy chain CDR interactions with the β-sheet surface of EGFR domain III that binds EGF. EGFR in tan with H1, H2, and H3 in cyan, gold and blue, respectively.
Fig 3.
Comparison of residue 468 binding sites.
(A) Binding site of panitumumab with EGFRD3 ND2. The S468 interaction with D100 is mediated through a water molecule. (B) Binding site of panitumumab with EGFRD3 ND2(S468R). R468 makes hydrogen bond interactions with D100, S440 and the main chain carbonyls of R101 and V102. (C) Binding site of cetuximab with S468 (PDB: 1YY9). Y104 packs against the S468 position and would not tolerate a large side chain in that position. (D) Model of EGFR S468R bound to cetuximab. The EGFR S468R mutation would severely clash with Y104. Model created by overlaying EGFRD3 ND2(S468R) onto EGFR domain III in the cetuximab structure without further refinement.
Fig 4.
(A) Panitumumab epitope on EGFR domain III of EGFR is shown in yellow. EGF binding surface on EGFR domain III is shown in red (PDB:1IVO). Mutations are shown on surface relative to panitumumab epitope and EGF binding interface. EGF and panitumumab have significant overlap in the domain III binding surfaces. S468R as well as many additional mutations are located on the perimeter of the EGF binding site but are centrally located in the panitumumab epitope. (B-E) Panitumumab is shown in magenta and cetuximab is shown in cyan. Red lettering represents mutation and the number in parenthesis represents numbering based on mature protein sequence used in the crystal structure. The remaining residues are numbered to be consistent with crystal structure numbering. (B)The K467T mutation will result in a clash with W94 in cetuximab but rotamer conformations exist that would minimize the steric clash in panitumumab, as a result cetuximab is sensitive to this mutation but panitumumab remains active. [13]. (C) I491M would result in steric clashes with D103 and Y102 in cetuximab and F91 and the main chain of T103 in panitumumab. (D) G465R would result in steric clashes with W52 and His35 in cetuximab and H52 and Y35 in panitumumab. (E) S464L is in a tightly packed region and would clash with Y102 and Y104 in cetuximab and V102 and Y35 in panitumumab.