Designed Asymmetric Protein Assembly on a Symmetric Scaffold

Lenne J.M. Lemmens; Job A.L. Roodhuizen; Tom F.A. de Greef; Albert J. Markvoort; Luc Brunsveld

doi:10.1002/ange.202003626

Designed Asymmetric Protein Assembly on a Symmetric Scaffold

Lenne J.M. Lemmens
, Job A.L. Roodhuizen
, Tom F.A. de Greef (Corresponding author)
, Albert J. Markvoort (Corresponding author)
, Luc Brunsveld (Corresponding author)

Chemical Biology

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

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Abstract

Cellular signaling is regulated by the assembly of proteins into higher-order complexes. Bottom-up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild-type symmetric CT32-CT32 interface of the 14-3-3-CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14-3-3 scaffold. Biochemical studies, supported by mass-balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher-order protein complexes. Enzyme complementation on the 14-3-3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.

Original language	English
Pages (from-to)	12211–12219
Number of pages	9
Journal	Angewandte Chemie
Volume	132
Issue number	29
DOIs	https://doi.org/10.1002/ange.202003626
Publication status	Published - 13 Jul 2020

Bibliographical note

Funding Information:
We thank Dr. Christian Ottmann for fruitful discussions and Dr. Stijn Aper for providing T14-3-3. This work was supported by The Netherlands Organization for Scientific Research (NWO) through the VICI grant 016.150.366 and VIDI grant 723.016.003, by the Ministry of Education, Culture and Science (Gravity program 024.001.035), and the European Research Council (project n. 677313 BioCircuit).

Funding

We thank Dr. Christian Ottmann for fruitful discussions and Dr. Stijn Aper for providing T14-3-3. This work was supported by The Netherlands Organization for Scientific Research (NWO) through the VICI grant 016.150.366 and VIDI grant 723.016.003, by the Ministry of Education, Culture and Science (Gravity program 024.001.035), and the European Research Council (project n. 677313 BioCircuit).

Keywords

cooperativity
noncovalent interactions
proteins
protein–protein interactions
self-assembly
Thermodynamics
Proteins/chemistry
Models, Chemical
Models, Molecular
Protein Binding
14-3-3 Proteins/chemistry
Protein Conformation

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title = "Designed Asymmetric Protein Assembly on a Symmetric Scaffold",

abstract = "Cellular signaling is regulated by the assembly of proteins into higher-order complexes. Bottom-up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild-type symmetric CT32-CT32 interface of the 14-3-3-CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14-3-3 scaffold. Biochemical studies, supported by mass-balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher-order protein complexes. Enzyme complementation on the 14-3-3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.",

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note = "Funding Information: We thank Dr. Christian Ottmann for fruitful discussions and Dr. Stijn Aper for providing T14-3-3. This work was supported by The Netherlands Organization for Scientific Research (NWO) through the VICI grant 016.150.366 and VIDI grant 723.016.003, by the Ministry of Education, Culture and Science (Gravity program 024.001.035), and the European Research Council (project n. 677313 BioCircuit). ",

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AU - Markvoort, Albert J.

AU - Brunsveld, Luc

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N2 - Cellular signaling is regulated by the assembly of proteins into higher-order complexes. Bottom-up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild-type symmetric CT32-CT32 interface of the 14-3-3-CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14-3-3 scaffold. Biochemical studies, supported by mass-balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher-order protein complexes. Enzyme complementation on the 14-3-3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.

AB - Cellular signaling is regulated by the assembly of proteins into higher-order complexes. Bottom-up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild-type symmetric CT32-CT32 interface of the 14-3-3-CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14-3-3 scaffold. Biochemical studies, supported by mass-balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher-order protein complexes. Enzyme complementation on the 14-3-3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.

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JO - Angewandte Chemie

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ER -

Designed Asymmetric Protein Assembly on a Symmetric Scaffold

Abstract

Bibliographical note

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Keywords

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