Fig. 1: Computational rational AAV capsid design to bind to αVβ6 integrin.

A Overview of the design pipeline, including three steps: 1. Capsid 3D structures were obtained either from the PDB database or predicted by AlphaFold2. 2. The capsid VR4 loop was completely replaced by integrating the binding motif, which was extracted from receptor’s natural binder, using RosettaRemodel protocol. 3. Top scored designs from the previous grafting step were docked onto the intended receptor in silico to verify the binding potential of the designed capsid. B An illustration of the sampling for low-energy sequence-structure pairs during motif-grafting process. Capsid VR4 after removing the loop was colored in blue, extracted binding motif was colored in red. The sampled linkers and sequences (Fig. S1F) were labeled in green. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). C, D The three lowest energy designs after grafting TGFβ3 (C) and TGFβ1 (D) into the capsid VR4. All top designs showed convergence in structures and sequences, suggesting sampling approached the global optimum. E, F Retrospective docking of motif-grafted capsids (E Cap9rh74_4um9) and (F Cap9rh74_5ffo) onto the αVβ6 structure. The left panels are illustrations of the structures with the lowest energy at the interface of capsid and integrin proteins (dG_separated: difference in free energy of two proteins). Both two newly designed VR4s (colored in green) were predicted to bind to the αVβ6 complex at very similar positions to natural binding motifs (colored in red). The right panels are scatter plots of dG_separated energy versus root-mean-square deviation (RMSD) from the lowest energy structure of all sampled docking positions.