Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Alena Khmelinskaia; Neville P Bethel; Farzad Fatehi; Bhoomika Basu Mallik; Aleksandar Antanasijevic; Andrew J Borst; Szu-Hsueh Lai; Ho Yeung Chim; Jing Yang 'John' Wang; Marcos C Miranda; Andrew M Watkins; Cassandra Ogohara; Shane Caldwell; Mengyu Wu; Albert J R Heck; David Veesler; Andrew B Ward; David Baker; Reidun Twarock; Neil P King

doi:10.1038/s41594-025-01490-z

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Alena Khmelinskaia^*, Neville P Bethel, Farzad Fatehi, Bhoomika Basu Mallik, Aleksandar Antanasijevic, Andrew J Borst, Szu-Hsueh Lai, Ho Yeung Chim, Jing Yang 'John' Wang, Marcos C Miranda, Andrew M Watkins, Cassandra Ogohara, Shane Caldwell, Mengyu Wu, Albert J R Heck, David Veesler, Andrew B Ward, David Baker, Reidun Twarock, Neil P King^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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
Article number	746
Journal	Nature Structural and Molecular Biology
DOIs	https://doi.org/10.1038/s41594-025-01490-z
Publication status	E-pub ahead of print - 26 Feb 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 Laender 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
Bill & Melinda Gates Foundation
Collaboration for AIDS Vaccine Discovery (CAVD)
National Science Foundation	DMREF 1629214
National Institute of Allergy and Infectious Disease	U54AI170856, 1P01AI167966, DP1AI158186, 75N93022C00036
Defense Threat Reduction Agency	HDTRA1-18-1-0001
Netherlands Organization for Scientific Research (NWO)	ENPPS.LIFT.019.001
Spinoza Award	SPI.2017.028
Wellcome Trust	110145, 110146
Krim Fellowship in Biomedical Research	110182-69-RKVA
HHMI Hanna Gray Fellowship
National Institute of Health
Royal Society Wolfson Fellowship
EPSRC Established Career Fellowship	EP/R023204/1
Wellcome Trust	110145, 110146
Not added	INV-010680
Not added	INV-002916
Not added	S10OD032290
Not added	RSWF/R1/180009

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10.1038/s41594-025-01490-zLicence: CC BY-NC-ND

Cite this

Khmelinskaia, A., Bethel, N. P., Fatehi, F., Mallik, B. B., Antanasijevic, A., Borst, A. J., Lai, S.-H., Chim, H. Y., Wang, J. Y. J., Miranda, M. C., Watkins, A. M., Ogohara, C., Caldwell, S., Wu, M., Heck, A. J. R., Veesler, D., Ward, A. B., Baker, D., Twarock, R., & King, N. P. (2025). Local structural flexibility drives oligomorphism in computationally designed protein assemblies. Nature Structural and Molecular Biology, Article 746. Advance online publication. https://doi.org/10.1038/s41594-025-01490-z

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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.",

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doi = "10.1038/s41594-025-01490-z",

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Khmelinskaia, A, Bethel, NP, Fatehi, F, Mallik, BB, Antanasijevic, A, Borst, AJ, Lai, S-H, Chim, HY, Wang, JYJ, Miranda, MC, Watkins, AM, Ogohara, C, Caldwell, S, Wu, M, Heck, AJR, Veesler, D, Ward, AB, Baker, D, Twarock, R & King, NP 2025, 'Local structural flexibility drives oligomorphism in computationally designed protein assemblies', Nature Structural and Molecular Biology. https://doi.org/10.1038/s41594-025-01490-z

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T1 - Local structural flexibility drives oligomorphism in computationally designed protein assemblies

AU - Khmelinskaia, Alena

AU - Bethel, Neville P

AU - Fatehi, Farzad

AU - Mallik, Bhoomika Basu

AU - Antanasijevic, Aleksandar

AU - Borst, Andrew J

AU - Lai, Szu-Hsueh

AU - Chim, Ho Yeung

AU - Wang, Jing Yang 'John'

AU - Miranda, Marcos C

AU - Watkins, Andrew M

AU - Ogohara, Cassandra

AU - Caldwell, Shane

AU - Wu, Mengyu

AU - Heck, Albert J R

AU - Veesler, David

AU - Ward, Andrew B

AU - Baker, David

AU - Twarock, Reidun

AU - King, Neil P

PY - 2025/2/26

Y1 - 2025/2/26

N2 - 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.

AB - 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.

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JO - Nature Structural and Molecular Biology

JF - Nature Structural and Molecular Biology

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ER -

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Abstract

Bibliographical note

Funding

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