Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

  • Nature Biotechnology volume 35, pages 667671 (2017)
  • doi:10.1038/nbt.3907
  • Download Citation


Many viral surface glycoproteins and cell surface receptors are homo-oligomers1,2,3,4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5,6,7,8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

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This work was funded by DTRA grants HDTRA1-16-C-0029 and HDTRA1-11-1-0041 (D.B), Life Science Discovery Fund Grant # 9598385 (D.B.), NIH 5R01AI096184-05 (P.Y.) and the NIH/NIAID R21AI119258 (D.H.F.). E.M.S. was supported by a career development award by the NW regional center of excellence (NIAID). J.D.B. and K.A.H. were supported by NIH grant R01 GM102198, K.A.H. by an NRSA training grant (T32GM007270) and I.A.W. and S.M.B. by R56 AI117675 and the Skaggs Institute for Chemical Biology at TSRI. A.J.B. was supported by a grant from the National Science Foundation (DGE-1256082). We would like to thank Erika O. Saphire for the generous gift of Ebola GP. We thank L. Carter and her team for help with protein purifications, Y. Song for help with comparative modeling and K. Godin for help with the selections and library generation. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

Author information


  1. Department of Biochemistry, University of Washington, Seattle, Washington, USA.

    • Eva-Maria Strauch
    • , David La
    • , Jorgen W Nelson
    • , William Sheffler
    •  & David Baker
  2. Institute for Protein Design, Department of Biochemistry, University of Washington, Seattle, Washington, USA.

    • Eva-Maria Strauch
    • , Rashmi Ravichandran
    •  & David Baker
  3. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Steffen M Bernard
    • , Peter S Lee
    • , Travis Nieusma
    • , Andrew B Ward
    •  & Ian A Wilson
  4. Department of Microbiology, University of Washington, Seattle, Washington, USA.

    • Alan J Bohn
    •  & Deborah H Fuller
  5. Department of Bioengineering, University of Washington, Seattle, Washington, USA.

    • Caitlin E Anderson
    • , Carly A Holstein
    •  & Paul Yager
  6. Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.

    • Natalie K Garcia
    •  & Kelly K Lee
  7. Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Kathryn A Hooper
    •  & Jesse D Bloom
  8. Washington National Primate Research Center, University of Washington, Seattle, Washington, USA.

    • Deborah H Fuller
  9. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.

    • David Baker


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E.-M.S., D.L. and W.S. designed proteins; D.L. designed the first version of the HSB protein, W.S. set-up the design pipeline for oligomerization, E.-M.S. designed the trimerization of the binders. E.-M.S. characterized designs, performed re-design of HSB, generated HA sequence alignments, homology models, library designs and selections; E.-M.S. and J.W.N. performed selection at different temperature and analyzed the deep-sequencing data. E.-M.S. and R.R. screened variants for higher expression and solubility. S.M.B. and E.-M.S. performed affinity measurements via biolayer interferometry. N.K.G. collected SAXS and HDX data, and analyzed it with K.K.L.; P.S.L. and S.M.B. expressed, purified and biotinylated HA proteins, P.S.L. crystallized HSB.2, S.M.B. crystallized the structure of the complex of HSB.2A with HA, P.S.L., S.M.B. and I.A.W. analyzed the crystallographic data. T.N. characterized the designed materials by electron microscopy; T.N. and A.B.W. analyzed electron microscopy data. K.A.H. and J.D.B. planned and performed the cell-based neutralization assay; C.E.A., C.A.H. and P.Y. designed and executed the paper-based diagnostic assays. A.J.B. and D.H.F. planned and performed the animal studies. E.-M.S. and D.B. analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The University of Washington has filed patents on the binding proteins.

Corresponding author

Correspondence to David Baker.

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