De novo design of high-affinity miniprotein binders targeting virulence factor.

poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella-like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well-defined binding pockets and structural information on native interactions has hindered structure-guided ligand discovery against Flpp3. Here, we used a combination of physics-based and deep-learning methods to design high-affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24-110 nM. For the second site, an initial binder showed a dissociation constant ( ) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub-nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X-ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (Cα root-mean-square deviation: 0.9 Å). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.