Abstract
Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, they primarily focus on designing static structures. Here we characterize three distinct computationally designed protein assemblies that exhibit unanticipated structural diversity arising from flexibility in their subunits. Cryo-EM single-particle reconstructions and native mass spectrometry reveal two distinct architectures for two assemblies, while six cryo-EM reconstructions for the third likely represent a subset of its solution-phase structures. Structural modeling and molecular dynamics simulations indicate that constrained flexibility within the subunits of each assembly promotes a defined range of architectures rather than nonspecific aggregation. Redesigning the flexible region in one building block rescues the intended monomorphic assembly. These findings highlight structural flexibility as a powerful design principle, enabling exploration of new structural and functional spaces in protein assembly design.
| Original language | English |
|---|---|
| Pages (from-to) | 1050-1060 |
| Number of pages | 11 |
| Journal | Nature Structural and Molecular Biology |
| Volume | 32 |
| Issue number | 6 |
| Early online date | 26 Feb 2025 |
| DOIs | |
| Publication status | Published - Jun 2025 |
Bibliographical note
Publisher Copyright:© The Author(s) 2025.
Funding
This work was funded by the Bill & Melinda Gates Foundation and the Collaboration for AIDS Vaccine Discovery (CAVD) (grant nos. INV-010680 to D.B. and N.P.K. and INV-002916 to A.B.W.), the National Science Foundation (grant nos. DMREF 1629214 to D.B. and N.P.K.), the National Institute of Allergy and Infectious Disease (grant nos. U54AI170856 to N.P.K., 1P01AI167966 to D.V. and N.P.K., DP1AI158186 to D.V., and 75N93022C00036 to D.V.), the Defense Threat Reduction Agency (grant no. HDTRA1-18-1-0001 to D.B. and N.P.K.), Netherlands Organization for Scientific Research (NWO) (grant no. ENPPS.LIFT.019.001 to A.J.R.H. and S.-H.L.), the Spinoza Award (grant no. SPI.2017.028 to A.J.R.H.), the Wellcome Trust (Joint Investigator Award nos. 110145 and 110146 to R.T.), an EPSRC Established Career Fellowship (grant no. EP/R023204/1 to R.T., which also provided funding for F.F.), a Royal Society Wolfson Fellowship (grant no. RSWF/R1/180009 to R.T.), generous gifts from the Audacious project and Open Philanthropy (D.B. and N.P.K.), the German Research Foundation (DFG) through the Collaborative Research Center SFB1032 (A.K.) and the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the L\u00E4nder through the ONE MUNICH Project Munich Multiscale Biofabrication (A.K., which also provided funding for H.Y.C). A.A. was supported by an amfAR Mathilde Krim Fellowship in Biomedical Research (grant no. 110182-69-RKVA), N.P.B. was supported by an HHMI Hanna Gray Fellowship (grant no. GT11817). Structural studies at the University of Washington were supported by the University of Washington Arnold and Mabel Beckman cryo-EM center and the National Institute of Health grant no. S10OD032290. D.V. and D.B. are Investigators of the Howard Hughes Medical Institute. We thank I. C. Haydon for support with graphical design. We thank D. Demurtas and M. Cantoni from the Center for Electron Microscopy center at EPFL for their assistance with the collection of nsEM data, the laboratory of O. Merkel for access to DLS instrumentation, P. Kielkowski for the quality control MS measurements of new designs, the Leibniz Supercomputing center for providing computing time on its Linux-Cluster and AI Systems, and the Khmelinskaia laboratory for essential laboratory support.
| Funders | Funder number |
|---|---|
| Bundesministerium für Bildung und Forschung | |
| Deutsche Forschungsgemeinschaft | |
| California Department of Fish and Game | |
| University of Washington Arnold and Mabel Beckman cryo-EM center | |
| Bill and Melinda Gates Foundation | |
| Mathilde Krim Fellowship in Biomedical Research | |
| Howard Hughes Medical Institute | GT11817 |
| Wellcome Trust | 110145, 110146 |
| Defense Threat Reduction Agency | HDTRA1-18-1-0001 |
| National Science Foundation | DMREF 1629214 |
| Collaboration for AIDS Vaccine Discovery | INV-010680, INV-002916 |
| amfAR, The Foundation for AIDS Research | 110182-69-RKVA |
| Engineering and Physical Sciences Research Council | EP/R023204/1 |
| Royal Society | RSWF/R1/180009 |
| Nederlandse Organisatie voor Wetenschappelijk Onderzoek | ENPPS.LIFT.019.001 |
| National Institute of Allergy and Infectious Diseases | U54AI170856, 1P01AI167966, DP1AI158186, 75N93022C00036 |
| National Institutes of Health | S10OD032290 |
| Collaborative Research Center | SFB1032 |