Fcab-HER2 Interaction: a Ménage à Trois. Lessons from X-Ray and Solution Studies

• DOI: 10.1016/j.str.2017.04.014
• Publication Date: 2017-05-18T18:32:15Z
• Authors: Elisabeth Lobner; Anne-Sophie Humm; Kathrin Göritzer; Georg Mlynek; Martin G. Puchinger; Christoph Hasenhindl; Florian Rüker; Michael W. Traxlmayr; Kristina Djinović...; Christian Obinger
• Keywords: Fcab-HER2 structure; PROTEINS; Protein Conformation; Receptor, ErbB-2; DIRECTED EVOLUTION; fluorescence correlation spectroscopy; Calorimetry; 106041 Strukturbiologie; Antibodies, Monoclonal, Humanized; Crystallography, X-Ray; VALIDATION; 03 medical and health sciences; ErbB2; Protein Domains; HER2; BINDING; EXTRACELLULAR REGION; Humans; HETEROGENEITY; X-ray crystallography; 0303 health sciences; IgG1-Fc; Binding Sites; STABILITY; REFINEMENT; FC FRAGMENT FCAB; Protein Stability; binding stoichiometry; 106041 Structural biology; Trastuzumab; Immunoglobulin Fc Fragments; 3. Good health; Spectrometry, Fluorescence; Fcab; Immunoglobulin G; Mutation; Chromatography, Gel; IGG1-FC

The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation of the overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.