Anticalins Reveal High Plasticity in the Mode of Complex Formation with a Common Tumor Antigen  André Schiefner, Michaela Gebauer, Antonia Richter, Arne Skerra  Structure  Volume 26, Issue 4, Pages 649-656.e3 (April 2018) DOI: 10.1016/j.str.2018.02.003 Copyright © 2018 Elsevier Ltd Terms and Conditions

Structure 2018 26, 649-656.e3DOI: (10.1016/j.str.2018.02.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Structural Overview of the Three ED-B⋅Anticalin Complexes: N7A⋅Fn7B8, N7E⋅Fn7B8, and N9B⋅ED-B (A) ED-B domain (in context of its two neighboring Fn domains, if present) arranged in the same orientation. (B) Close-up view of the Anticalin⋅ED-B interfaces with the Anticalins depicted in equivalent orientations. Structure 2018 26, 649-656.e3DOI: (10.1016/j.str.2018.02.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 Comparison of Anticalin Contacts with the Bound Fn Fragment (A) Amino acid sequence alignment of the three Anticalins N7A, N7E, and N9B using ALINE (Bond and Schüttelkopf, 2009). Residues that interact with ED-B or Fn8 in each complex are highlighted in the same colors as for the Anticalins depicted in Figure 1 while randomized positions are indicated with asterisks (Gebauer et al., 2013). (B) Structural superposition of wild-type Lcn2 (PDB: 1L6M) (Goetz et al., 2002) and the three Anticalins, N7A, N7E, and N9B (cf. Supplemental Information). The structurally variable loops #1 to #4 are colored as in Figure 1, whereas those of wild-type Lcn2 are colored blue. (C) Surface representation of the Anticalins N7A, N7E, and N9B in the same orientation as in (B), right. Target contact residues are colored according to their side-chain properties: acidic (red), basic (blue), polar uncharged (green), and hydrophobic (brown). Structure 2018 26, 649-656.e3DOI: (10.1016/j.str.2018.02.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 Contact Analysis of Fn Fragments Bound to the Three Different Anticalins (A) Surface representation of Fn with ED-B (dark gray) and Fn8 (light gray). The contact surfaces are highlighted in the same color as for the Anticalins shown in Figure 1. (B) Recognition of the ED-B cc' loop in highly individual orientations and conformations by the Anticalins N7A, N7E, and N9B (light gray surface). The cc' loop is shown as ribbon in the same color as in (A). Interacting residues of each Anticalin are shown in dark gray. In both (A and B) the hydrogen bond as well as the salt bridge donors and acceptors are highlighted in blue and red, respectively, indicating that the Fn target molecules mainly act as acceptors. Structure 2018 26, 649-656.e3DOI: (10.1016/j.str.2018.02.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 Conformation of the FnB8 Epitope in Complex with the Anticalins N7A or N7E Compared with the Uncomplexed Fn7B89 Homodimer Superposition of ED-B as part of the FnB8 moieties as observed, respectively, in the complex with N7A (left, violet; PDB: 4GH7) and N7E (right, orange; PDB: 5N47) on the (uncomplexed) Fn7B89 dimer (gray; PDB: 3T1W), thus illustrating the relative domain orientations of Fn8. The green circle highlights the sterical accessibility of the ED-B cc' loop in the context of the assembled Fn dimer. Structure 2018 26, 649-656.e3DOI: (10.1016/j.str.2018.02.003) Copyright © 2018 Elsevier Ltd Terms and Conditions