De Novo Design of High‐Affinity Miniprotein Binders Targeting <i>Francisella Tularensis</i> Virulence Factor
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<sub>D</sub>) 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.
Authors
- Yensi Flores Bueso (ORCID: https://orcid.org/0000-0002-2118-2195)
- Stacey Gerben (ORCID: https://orcid.org/0000-0003-0313-6248)
- Asim K. Bera (ORCID: https://orcid.org/0000-0001-9473-2912)
- Buwei Huang (ORCID: https://orcid.org/0000-0002-0762-7178)
- Dionne Vafeados (ORCID: https://orcid.org/0000-0002-2560-8907)
- Joseph D. Mougous (ORCID: https://orcid.org/0000-0002-5417-4861)
- Stephen Rettie (ORCID: https://orcid.org/0000-0001-9797-6939)
- Alex Kang (ORCID: https://orcid.org/0000-0001-5487-0499)
- Andi Liu (ORCID: https://orcid.org/0000-0002-8795-9614)
- Gaurav Bhardwaj (ORCID: https://orcid.org/0000-0001-6554-2335)
- David A. Baker (ORCID: https://orcid.org/0000-0001-7896-6217)
- Inna Goreshnik (ORCID: https://orcid.org/0000-0002-9254-9249)
- Victor Adebomi (ORCID: https://orcid.org/0000-0002-4573-5102)
- Xinting Li (ORCID: https://orcid.org/0000-0001-5559-7578)
- Joshua J. Woodward (ORCID: https://orcid.org/0000-0002-4630-403X)
- Lieselotte S.M. Kreuk (ORCID: https://orcid.org/0000-0003-4906-039X)
- Gizem Gokce‐Alpkilic (ORCID: https://orcid.org/0000-0001-5009-4429)
- Yaxi Wang (ORCID: https://orcid.org/0000-0003-3278-6127)
- Nicole Roullier
Institutions
- Howard Hughes Medical Institute (US)
- University of Washington (US)
- University College Cork (IE)
- Zero to Three (US)
- PDL BioPharma (United States) (US)
- Ecologie microbienne (FR)
Publication Details
- Journal
- Angewandte Chemie International Edition
- Published
- 2025-10-21
- DOI
- https://doi.org/10.1002/anie.202516058
- Primary Topic
- Bacillus and Francisella bacterial research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Howard Hughes Medical Institute
- U.S. Department of Energy
- University of Washington
- National Institutes of Health
- Defense Advanced Research Projects Agency
- Office of Science
- National Institute of General Medical Sciences
- Biological and Environmental Research
- H2020 Marie Skłodowska-Curie Actions