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Designed Asymmetric Protein Assembly on a Symmetric Scaffold
Author(s) -
Lemmens Lenne J. M.,
Roodhuizen Job A. L.,
Greef Tom F. A.,
Markvoort Albert J.,
Brunsveld Luc
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202003626
Subject(s) - cooperativity , scaffold , scaffold protein , ternary complex , ternary operation , chemistry , affinities , protein engineering , protein design , complementation , protein structure , nanotechnology , biophysics , stereochemistry , enzyme , materials science , biochemistry , computer science , biology , signal transduction , mutant , database , programming language , gene
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.