Monobodies and other synthetic binding proteins for expanding protein science

Sha, Fern; Salzman, Gabriel S.; Gupta, Ankit; Koide, Shohei

doi:10.1002/pro.3148

Cited by 145 publications

(127 citation statements)

References 80 publications

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“…Similarly, BCL-2 binding Affimers themselves could be elaborated for therapeutic or diagnostic use. 34,66 Of potentially equal significance is the observation that non-canonical folds can substitute for native folds in peptide/protein based inhibitors of PPIs 67,68 ; this is reminiscent of the use of a β-hairpin to mimic an α-helix for p53/hDM2 inhibition. 69,70 In contrast to those studies, the sequences identified here were obtained under selection pressure, and this poses the question: do BCL-2 family proteins function in cells through molecular modes of interaction other than the canonical α-helix/cleft motif observed to date?…”

Section: Discussion (Without Subheadings)mentioning

confidence: 99%

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Miles¹,

Hóbor²,

Taylor³

et al. 2019

Preprint

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no more than 200 words)The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Cocrystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-ofconcept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications. [198 words]

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Section: Discussion (Without Subheadings)mentioning

confidence: 99%

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Miles¹,

Hóbor²,

Taylor³

et al. 2019

Preprint

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“…Thus, many positions of the frankenbody can be mutated without destroying its functionality, demonstrating great potential for further evolution. We therefore anticipate the HA frankenbody will be a useful new imaging reagent to complement the growing arsenal of live-cell antibody-based probes 23,[49][50][51] .…”

Section: Discussionmentioning

confidence: 99%

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

Zhao

Kamijo

Fox

et al. 2018

Preprint

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One-sentence summary:A genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics. ABSTRACTTo expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art singlemolecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful new tool for imaging protein dynamics in vivo.

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“…We have achieved success with a number of different formats including scFv intrabodies, FN3s, and DARPins. Other scaffolds may also work, provided that they are small, fold efficiently in the reducing cytosolic environment, and exhibit high affinity (low µM to nM) and specificity towards the intended target (Sha, Salzman, Gupta, & Koide, 2017). There have been several reports describing successful fusions between DBPs and truncated E3 ubiquitin ligases (Caussinus et al, 2011; Portnoff et al, 2014; Shin et al, 2015).…”

Section: Strategic Planningmentioning

confidence: 99%

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion

Baltz

Stephens

DeLisa

2018

CP Chemical Biology

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A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called "ubiquibodies," are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity-simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active versus inactive conformation, mutant versus wild-type, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins. © 2018 by John Wiley & Sons, Inc.

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Monobodies and other synthetic binding proteins for expanding protein science

Cited by 145 publications

References 80 publications

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion

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