De novo design of obligate ABC-type heterotrimeric proteins

Sherry Bermeo; Andrew Favor; Ya-Ting Chang; Andrew Norris; Scott E Boyken; Yang Hsia; Hugh K Haddox; Chunfu Xu; T J Brunette; Vicki H Wysocki; Gira Bhabha; Damian C Ekiert; David Baker

doi:10.1038/s41594-022-00879-4

De novo design of obligate ABC-type heterotrimeric proteins

Nat Struct Mol Biol. 2022 Dec;29(12):1266-1276. doi: 10.1038/s41594-022-00879-4. Epub 2022 Dec 15.

Authors

Sherry Bermeo^#^{1

2

3}, Andrew Favor^#^{1

2

4}, Ya-Ting Chang^#⁵, Andrew Norris^{6

7}, Scott E Boyken^{1

2}, Yang Hsia^{1

2}, Hugh K Haddox^{1

2}, Chunfu Xu^{1

2

8}, T J Brunette^{1

2}, Vicki H Wysocki^{6

7}, Gira Bhabha⁵, Damian C Ekiert^{5

9}, David Baker^{10

11

12}

Affiliations

¹ Department of Biochemistry, University of Washington, Seattle, WA, USA.
² Institute for Protein Design, University of Washington, Seattle, WA, USA.
³ Biological Physics, Structure and Design Graduate Program, University of Washington, Seattle, WA, USA.
⁴ Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, USA.
⁵ Department of Cell Biology, New York University School of Medicine, New York, NY, USA.
⁶ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
⁷ Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH, USA.
⁸ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
⁹ Department of Microbiology, New York University School of Medicine, New York, NY, USA.
¹⁰ Department of Biochemistry, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
¹¹ Institute for Protein Design, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
¹² Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. dabaker@uw.edu.

^# Contributed equally.

Abstract

The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Models, Molecular
Proteins* / chemistry

Substances

Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding