Structural basis of Fusobacterium nucleatum adhesin Fap2 interaction with receptors on cancer and immune cells

The intestinal microbiome (IM) is decisive for the human hosts health. Numerous microbiota drive the progression of colorectal cancer (CRC), the third-most common cancer worldwide. The Gram-negative Fusobacterium nucleatum (Fn) is overrepresented in the IM of CRC patients and has been correlated with the emergence, progression, and metastasis of tumors. A key pathogenic factor of Fn is the adhesin Fap2, an autotransporter protein that facilitates association to cancer and immune cells via two receptors, the glycan Gal-GalNAc and the T-cell protein TIGIT, respectively. The latter interaction leads to deactivation of immune cells. Mechanistic details of the Fap2/TIGIT interaction remain elusive due to the lack of high-resolution structural data. Here, we report a system to recombinantly express functional Fap2 on the Escherichia coli surface, which interacts with Gal-GalNAc on cancer cells and with purified TIGIT with submicromolar affinity. Cryo-EM structures of Fap2, alone and in complex with TIGIT, show that the [~]50 nm long rod-shaped Fap2 extracellular region binds to TIGIT on its membrane-distal tip via an extension of a {beta}-helix domain. Moreover, by combining structure predictions, cryo-EM, docking and MD simulations, we identified a binding pit for Gal-GalNAc on the tip of Fap2. Our data represent the first purification and high-resolution structural analysis of a Fn autotransporter adhesin and its receptor association.