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Published June 8, 2021 | Published + Supplemental Material + Submitted
Journal Article Open

Generation of ordered protein assemblies using rigid three-body fusion

Abstract

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Additional Information

© 2021 National Academy of Sciences. Published under the PNAS license. Edited by Shane Gonen, University of California, Irvine, CA, and accepted by Editorial Board Member William F. DeGrado April 18, 2021 (received for review July 21, 2020). Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) under the NIH under Award Number T32GM008268 to I.V., and the Open Philanthropy Project, HHMI, and NSF grant CHE-1629214 to D.B. NIH grant under award AI150464 provided support to G.J.J. Cryo-EM work under G.J.J. was performed in the Caltech Beckman Institute Resource Center for Transmission Electron Microscopy. We also thank Dr. Songye Chen and Dr. Andrey Malyutin at Caltech for technical assistance. This work was also supported by the National Institute of Allergy and Infectious Diseases (NIAID) grant DP1AI158186, NIH grant HHSN272201700059C, NIGMS grant R01GM120553, a Pew Biomedical Scholars Award, and a Burroughs Wellcome Investigators in the Pathogenesis of Infectious Diseases award to D.V. This work was also supported by NIH grant P41GM128577 to V.H.W. and Swiss National Science Foundation grant 310030_192689 to A.P. In addition, we thank Kathryn Burnett and Greg Hura for SAXS data collection through the SIBYLS mail-in SAXS program at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies program, supported by the Department of Energy (DOE) Office of Biological and Environmental Research. Additional support comes from the NIH project ALS-ENABLE (grant P30 GM124169) and High-End Instrumentation Grant S10OD018483. A.C. is a recipient of the Human Frontiers Science Program Long Term Fellowship. A.C. and D.D.S. received Washington Research Foundation fellowships. We thank Albumedix for providing high-quality Veltis-grade HSA. I.V. thanks Shane Caldwell (University of Washington) for discussion on SAXS analysis and Vikram Mulligan (Flatiron Institute) for answering questions on RosettaScripts. Data Availability: Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (see SI Appendix for details), and the fusion method implementation is available on GitHub (archived with Zenodo at https://zenodo.org/record/4771121) (58). Additional supporting data is deposited with Zenodo (https://zenodo.org/record/4771103) (59) and all other data is included in the article or supporting information. Q.Y. and Y.-J.P. contributed equally to this work. Author contributions: I.V., Q.Y., G.J.J., and D.B. designed research; I.V. developed software; I.V., Q.Y., Y.-J.P., A.C., A.N., F.B., A.S., D.D.S., G.U., J.A.F., S.J.W., Y.H., V.H.W., D.V., and G.J.J. performed research; H.M., R.A.L., and A.P. contributed new reagents/analytic tools; I.V., Q.Y., Y.-J.P., A.C., A.N., F.B., A.S., and S.J.W. analyzed data; and I.V., Q.Y., Y.-J.P., A.C., A.N., F.B., H.M., D.D.S., A.P., V.H.W., D.V., G.J.J., and D.B. wrote the paper. The authors declare no competing interest. This article is a PNAS Direct Submission. S.G. is a guest editor invited by the Editorial Board. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2015037118/-/DCSupplemental.

Attached Files

Published - e2015037118.full.pdf

Submitted - 2020.07.18.210294v1.full.pdf

Supplemental Material - pnas.2015037118.sapp.pdf

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Additional details

Created:
August 22, 2023
Modified:
December 22, 2023