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De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor

Gokce‐Alpkilic, Gizem [Molecular Engineering and Sciences Institute University of Washington Seattle WA USA; Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA] (ORCID:0000000150094429)·Huang, Buwei [Institute for Protein Design University of Washington Seattle WA USA; Department of Biochemistry University of Washington Seattle WA USA; Department of Bioengineering University of Washington Seattle WA USA]·Liu, Andi [Department of Microbiology University of Washington Seattle WA USA]·Kreuk, Lieselotte S.M. [Department of Microbiology University of Washington Seattle WA USA]·Wang, Yaxi [Department of Microbiology University of Washington Seattle WA USA]·Adebomi, Victor [Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA]·Bueso, Yensi Flores [Institute for Protein Design University of Washington Seattle WA USA; Cancer Research @UCC University College Cork Cork Ireland]·Bera, Asim K. [Institute for Protein Design University of Washington Seattle WA USA]·Kang, Alex [Institute for Protein Design University of Washington Seattle WA USA]·Gerben, Stacey R. [Institute for Protein Design University of Washington Seattle WA USA]·Rettie, Stephen [Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA; Molecular and Cellular Biology Program University of Washington Seattle WA USA]·Vafeados, Dionne K. [Institute for Protein Design University of Washington Seattle WA USA]·Roullier, Nicole [Institute for Protein Design University of Washington Seattle WA USA]·Goreshnik, Inna [Institute for Protein Design University of Washington Seattle WA USA]·Li, Xinting [Institute for Protein Design University of Washington Seattle WA USA]·Baker, David [Institute for Protein Design University of Washington Seattle WA USA; Department of Biochemistry University of Washington Seattle WA USA; Howard Hughes Medical Institute University of Washington Seattle WA USA]·Woodward, Joshua J. [Department of Microbiology University of Washington Seattle WA USA]·Mougous, Joseph D. [Department of Microbiology University of Washington Seattle WA USA; Howard Hughes Medical Institute University of Washington Seattle WA USA; Microbial Interactions and Microbiome Center University of Washington Seattle WA USA]·Bhardwaj, Gaurav [Molecular Engineering and Sciences Institute University of Washington Seattle WA USA; Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA]
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Abstract

Abstract Francisella tularensis 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 ( K D ) 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 (RMSD): 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.

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BibTeXRIS

Gokce‐Alpkilic, Gizem [Molecular Engineering and Sciences Institute University of Washington Seattle WA USA; Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA] (ORCID:0000000150094429), Huang, Buwei [Institute for Protein Design University of Washington Seattle WA USA; Department of Biochemistry University of Washington Seattle WA USA; Department of Bioengineering University of Washington Seattle WA USA], Liu, Andi [Department of Microbiology University of Washington Seattle WA USA], Kreuk, Lieselotte S.M. [Department of Microbiology University of Washington Seattle WA USA], Wang, Yaxi [Department of Microbiology University of Washington Seattle WA USA], Adebomi, Victor [Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA], Bueso, Yensi Flores [Institute for Protein Design University of Washington Seattle WA USA; Cancer Research @UCC University College Cork Cork Ireland], Bera, Asim K. [Institute for Protein Design University of Washington Seattle WA USA], Kang, Alex [Institute for Protein Design University of Washington Seattle WA USA], Gerben, Stacey R. [Institute for Protein Design University of Washington Seattle WA USA], Rettie, Stephen [Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA; Molecular and Cellular Biology Program University of Washington Seattle WA USA], Vafeados, Dionne K. [Institute for Protein Design University of Washington Seattle WA USA], Roullier, Nicole [Institute for Protein Design University of Washington Seattle WA USA], Goreshnik, Inna [Institute for Protein Design University of Washington Seattle WA USA], Li, Xinting [Institute for Protein Design University of Washington Seattle WA USA], Baker, David [Institute for Protein Design University of Washington Seattle WA USA; Department of Biochemistry University of Washington Seattle WA USA; Howard Hughes Medical Institute University of Washington Seattle WA USA], Woodward, Joshua J. [Department of Microbiology University of Washington Seattle WA USA], Mougous, Joseph D. [Department of Microbiology University of Washington Seattle WA USA; Howard Hughes Medical Institute University of Washington Seattle WA USA; Microbial Interactions and Microbiome Center University of Washington Seattle WA USA], Bhardwaj, Gaurav [Molecular Engineering and Sciences Institute University of Washington Seattle WA USA; Department of Medicinal Chemistry University of Washington Seattle WA USA; Institute for Protein Design University of Washington Seattle WA USA]. 2025-10-21. De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor. https://doi.org/10.1002/anie.202516058

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