Figure 4.

Proposed binding mode of MEDI3622. (A) Three-dimensional model of MEDI3622 bound to TACE. The identified epitope of MEDI3622 (P³⁶⁶-N³⁸¹) (cyan) adopts a β-hairpin loop conformation projecting from the central body of M-domain (magenta). TACE residues Y³⁷⁰ and Y³⁷⁹ (cyan) along with conserved histidines H⁴⁰⁵, H⁴⁰⁹, H⁴¹⁵ (yellow) are shown in sticks. The Zn ion is shown as a green sphere. MEDI3622 (V_H in orange, V_L in beige) binds to TACE through interacting with the sIVa-sIVb β-hairpin loop, with the sIVa strand (Y³⁶⁹Y³⁷⁰S³⁷¹) located in the center of the binding interface. (B) Binding of MEDI3622 to TACE alanine mutants. Two stretches of amino acids were mutated into alanines, including Y³⁶⁹Y³⁷⁰S³⁷¹ (sIVa strand, the center of the binding interface) and N³⁷⁷I³⁷⁸Y³⁷⁹ (sIVb strand, peripheral to the binding interface). The alanine variants were expressed as soluble proteins and their binding profiles with MEDI3622 were characterized using SPR. Binding was calculated as % binding compared to wild type TACE after normalization of expression levels using the following formula: [(Response _{TACE variants MEDI3622}/Response _{TACE wildtype MEDI3622})/(Response _{TACE variants polyAb}/Response _{TACE wildtype polyAb})] *100. Binding of MEDI3622 to TACE was abolished when mutating residues Y³⁶⁹Y³⁷⁰S³⁷¹ to alanines, while its binding was retained upon mutating residues N³⁷⁷I³⁷⁸Y³⁷⁹. Results represent the means of 3 independent experiments with error bars indicating standard deviations.