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Cyclic oligomer design with de novo ??-proteins.

ABSTRACT: We have previously shown that monomeric globular ??-proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo-oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo-oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo-oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with ?-strand backbone interactions were structurally confirmed through x-ray crystallography and small-angle X-ray scattering (SAXS). In contrast, C3-C5 designed homo-oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo-oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.

SUBMITTER: Lin YR

PROVIDER: S-EPMC5654858 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Cyclic oligomer design with de novo αβ-proteins.

Lin Yu-Ru YR Koga Nobuyasu N Vorobiev Sergey M SM Baker David D

Protein science : a publication of the Protein Society 20171101 11

We have previously shown that monomeric globular αβ-proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo-oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo-oligomer. The latter flexible backbone protocol gener ...[more]

PMID: 28801928

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Project description:Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gα q and its effector PLC-β isozymes. Several of the designed proteins remain folded above 90°C and bind to Gα q with equilibrium dissociation constants tighter than 80 nM. In cellular assays with oncogenic variants of Gα q , the designed proteins inhibit activation of PLC-β isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection.statement for broader audienceEngineered proteins that bind to specific target proteins are useful as research reagents, diagnostics, and therapeutics. We used computational protein design to engineer de novo proteins that bind and competitively inhibit the G protein, Gα q , which is an oncogene for uveal melanomas. This computational method is a general approach that should be useful for designing competitive inhibitors against other proteins of interest.

Dataset Information

Cyclic oligomer design with de novo ??-proteins.

Publications

Cyclic oligomer design with de novo αβ-proteins.

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