Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang‐Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

doi:10.1038/ncomms11261

Cited by 56 publications

(53 citation statements)

References 56 publications

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“…Additionally, the approach does not rely on the ribosomal machinery and thus delivers a homogenous protein population by avoiding the potential for non-specific incorporation, which can affect protein manipulation using non-sense suppression approaches [32][33][34][35][36] .…”

Section: Discussionmentioning

confidence: 99%

“…This is particularly true for complex proteins expressed in eukaryotic cells. In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 36 , ion channels [37][38][39] , and G protein-coupled receptors 40,41 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]42 or by adding affinity tags to promote split intein interactions 18 .…”

Section: Discussionmentioning

confidence: 99%

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Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Khoo

Galleano

Gasparri

et al. 2020

Preprint

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AbstractManipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Khoo

Galleano

Gasparri

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…[22] hSERTregulates neuronal signalling by orchestrating serotonin metabolism. [22] hSERTregulates neuronal signalling by orchestrating serotonin metabolism.…”

Section: Mapping Receptor-ligand Interactions In Signal-transduction mentioning

confidence: 99%

“…In as imilar approach, Rannversson et al mapped the binding sites for two antidepressants (imipramine and vortioxetine) on the human serotonin receptor (hSERT). [22] hSERTregulates neuronal signalling by orchestrating serotonin metabolism. Inhibitors of hSERTare extensively used in the treatment of psychiatric diseases,s uch as depression and anxiety.I nm any cases,h owever,t he exact binding mode to the human receptor is not known in detail.…”

Section: Biological Applications 231 Mapping Receptor-ligand Intermentioning

confidence: 99%

Expanding the Genetic Code to Study Protein–Protein Interactions

2018

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Protein-protein interactions are central to many biological processes. A considerable challenge consists however in understanding and deciphering when and how proteins interact, and this can be particularly difficult when interactions are weak and transient. The site-specific incorporation of unnatural amino acids (UAAs) that crosslink with nearby molecules in response to light provides a powerful tool for mapping transient protein-protein interactions and for defining the structure and topology of protein complexes both in vitro and in vivo. Complementary strategies consist in site-specific incorporation of UAAs bearing electrophilic moieties that react with natural nucleophilic amino acids in a proximity-dependent manner, thereby chemically stabilizing low-affinity interactions and providing additional constraints on distances and geometries in protein complexes. Herein, we review how UAAs bearing fine-tuned chemical moieties that react with proteins in their vicinity can be utilized to map, study, and characterize weak and transient protein-protein interactions in living systems.

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“…-B)(32)(33)(34). Incorporating AzF at positions lining the ASIC1a-BigDyn interaction interface should allow covalent trapping of the complex and enable subsequent visualization using western blotting.…”

mentioning

confidence: 99%

Mechanism and site of action of big dynorphin on ASIC1a

Borg

Braun

Heusser

et al. 2019

Preprint

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Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, as well as initiation of pain and neuronal death following ischemic stroke.As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acidmediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Note: This manuscript has not been peer-reviewed 3

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Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

Cited by 56 publications

References 56 publications

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Expanding the Genetic Code to Study Protein–Protein Interactions

Mechanism and site of action of big dynorphin on ASIC1a

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