Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules

Rimbault, Charlotte; Maruthi, Kashyap; Breillat, Christelle; Genuer, Camille; Crespillo, Sara; Puente-Muñoz, Virginia; Chamma, Ingrid; Gauthereau, Isabel; Antoine, Ségolène; Thibaut, Coraline; Tai, Fabienne Wong Jun; Dartigues, Benjamin; Grillo-Bosch, Dolors; Claverol, Stéphane; Poujol, Christel; Choquet, Daniel; Mackereth, Cameron D.; Sainlos, Matthieu

doi:10.1038/s41467-019-12528-4

Cited by 27 publications

(52 citation statements)

References 80 publications

(102 reference statements)

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“…Using a phage display selection approach with a 10 FN3derived library, we have recently isolated and characterized three monobodies targeting PSD-95 (Rimbault et al, 2019). The clones were targeted against PSD-95 tandem PDZ domains and showed remarkable specificity for PSD-95, in particular when considering the high sequence conservation of paralogs (SAP97, SAP102 and PSD-93).…”

Section: Introductionmentioning

confidence: 99%

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Rimbault

Breillat

Compans

et al. 2021

Preprint

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Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

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Section: Introductionmentioning

confidence: 99%

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Rimbault

Breillat

Compans

et al. 2021

Preprint

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“…To verify γ2 S44* accumulation along the Z-projected dendritic shaft (Fig. 4d), we co-expressed γ2 S44* with the PSD-95 marker XPH20 fused with eGFP (XPH20::eGFP) 48 as a reporter and found that γ2 S44* accumulation was indeed always colocalized with the XPH20 eGFP signal (Fig. 4k).…”

Section: Resultsmentioning

confidence: 97%

“…The plasmid for the expression of the tRNA/aminoacyl transferase pair (pNEU-hMbPylRS- 4xU6M15, herein termed PylRS/4xtRNA Pyl ) was a gift from Irene Coin (Addgene, #105830) 44 . The plasmid for the expression of the Xph20 eGFP CCR5TC (XPH20::eGFP) was a gift from Matthieu Sainlos 48 .…”

Section: Methodsmentioning

confidence: 99%

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Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

Choquet

Sauer

Bessa-Neto

et al. 2021

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Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution, typically nowadays a few nanometers, and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

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“…[48][49][50] Ligand discovery for PDZ domains has proven challenging (e.g. fragment-based screening was unsuccessful for the PDZ domain of PSD-95 51 ), with the majority of potent ligands based on protein 52,53 , peptide 54,55 or peptidomimetic scaffolds 26,[56][57][58] and limited examples of small-molecules. [59][60][61][62] This rendered the GKAP/SHANK-PDZ interaction as a stringent test for our dynamic ligation screening approach.…”

Section: Introductionmentioning

confidence: 99%

Identification of β-Strand Mediated Protein-Protein Interaction Inhibitors Using Ligand-Directed Fragment Ligation

Hegedüs¹,

Hóbor²,

Shoemark³

et al. 2020

Preprint

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β-Strand mediated protein-protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and <i>in situ</i> screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.

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Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules

Cited by 27 publications

References 80 publications

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

Identification of β-Strand Mediated Protein-Protein Interaction Inhibitors Using Ligand-Directed Fragment Ligation

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