A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Jedlitzke, Benedikt; Mootz, Henning D.

doi:10.1002/cbic.202200079

Cited by 8 publications

(7 citation statements)

References 39 publications

(55 reference statements)

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“…First, NBY has been proposed as a universal proximal cage for the temporal blockage of protein activity (Wang et al, 2019;Wang et al, 2021). Second, NBY has been successfully employed to photocontrol antigen-antibody interactions (Bridge et al, 2019;Jedlitzke et al, 2019;Joest et al, 2021;Jedlitzke and Mootz, 2022;O'Shea et al, 2022;Yilmaz et al, 2022;Bridge et al, 2023).…”

Section: Introductionmentioning

confidence: 99%

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Pham

Zahradnı́k

Kolářová

et al. 2023

Front. Mol. Biosci.

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Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.

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Section: Introductionmentioning

confidence: 99%

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Pham

Zahradnı́k

Kolářová

et al. 2023

Front. Mol. Biosci.

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“…8,9 Alternatively, a collection of Nbs specifically raised against a prototypical, synthetic α-helical peptide (Alfa Tag) have been used for a variety of applications. 10,11 Nanobodies raised against the mammalian proteins α-synuclein, 12 β-catenin, 13 CXCR2 (refs 14, 15), and UBC6e 16 have been shown to bind to small peptide epitopes taken from these proteins. Previous work has shown that a nanobody (previously called VHH05 or VHH 6E , here named Nb 6E ) binds to a 14-mer peptide derived from the protein UBC6e (6E tag) with low nanomolar affinity.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Efforts have been undertaken to identify Nbs that bind to peptide epitope tags . Nbs that bound to the human immunodeficiency virus protein gp41 were shown to bind to short peptide fragments of this protein, which could be applied as epitope tags (MoonTag, PepTag). , Alternatively, a collection of Nbs specifically raised against a prototypical, synthetic α-helical peptide (Alfa Tag) have been used for a variety of applications. , Nanobodies raised against the mammalian proteins α-synuclein, β-catenin, CXCR2 (refs , ), and UBC6e have been shown to bind to small peptide epitopes taken from these proteins. Previous work has shown that a nanobody (previously called VHH05 or VHH 6E , here named Nb 6E ) binds to a 14-mer peptide derived from the protein UBC6e (6E tag) with low nanomolar affinity .…”

Section: Introductionmentioning

confidence: 99%

Characterization of a Nanobody-Epitope Tag Interaction and Its Application for Receptor Engineering

2022

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Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here, we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody–epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody–epitope pair opens the door to diverse applications including mechanistic studies of the G protein-coupled receptor function.

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“…However, this approach suffers from low selectivity and versatility. These problems have been overcome by genetic code expansion, which uses the reprogramming of a stop codon to incorporate ncAAs at practically any desired position of a protein of interest. − In this respect, photoxenoprotein engineering has enabled photocontrol of protein functions including modulation of enzyme activity and allostery, protein binding affinities, protein localization, and protein assembly and multimerization. − …”

Section: Introductionmentioning

confidence: 99%

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase

et al. 2022

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The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

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A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Cited by 8 publications

References 39 publications

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

Characterization of a Nanobody-Epitope Tag Interaction and Its Application for Receptor Engineering

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase

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