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The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53’s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with AI-based computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53’s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

We have developed a rapid and highly specific assay for detecting and monitoring SARS-CoV-2 infections by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As MALDI-TOF mass spectrometers are available in a clinical setting, our assay has the potential to serve as alternative to the commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sample preparation prior to MALDI-TOF-MS involves the tryptic digestion of SARS-CoV-2 proteins, followed by an enrichment of virus-specific peptides from SARS-CoV-2 nucleoprotein via magnetic antibody beads. Our MALDI-TOF-MS method allows the detection of SARS-CoV-2 nucleoprotein as low as 8 amol/microliter. MALDI-TOF mass spectra are obtained in just a few seconds, which makes our MS-based assay suitable for a high-throughput screening of SARS-CoV-2 in healthcare facilities in addition to PCR. Due to the specific detection of virus peptides, different SARS-CoV-2 variants are readily distinguished from each other. Specifically, we show that our MALDI-TOF-MS assay discriminates SARS-CoV-2 strain B.1.617.2 delta variant from all other variants in patients' samples, making our method highly valuable to monitor the emergence of new virus variants.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (i.e. trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to Down syndrome-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-Cas9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in ML-DS patients, and mechanistic studies using murine ML-DS models and patient-derived xenografts (PDXs) revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in PDXs in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong anti-leukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy bodies in neurons, which are the hallmark of Parkinson's Disease (PD). Neurotoxicity precedes the formation of aggregates and might be related to α-syn LLPS. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). In this work, we show that the conformational ensemble of α-syn shifts from a "hairpin-like" structure towards more "elongated" conformational states upon LLPS. We obtain insights into the critical initial stages of LLPS and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here, we explore the hypothesis that tumor cells express cancer-specific surface protein conformations, invisible to standard target discovery pipelines evaluating gene or protein expression, that can be identified and immunotherapeutically targeted. We term this strategy, integrating cross-linking mass spectrometry (XL-MS) with glycoprotein surface capture, “structural surfaceomics”. As a proof of principle, we apply this technology to acute myeloid leukemia, a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin-β2 as a structurally-defined, widely-expressed, AML-specific target. We develop and characterize recombinant antibodies to this protein conformation, and show that chimeric antigen receptor (CAR) T-cells eliminate AML cells and patient-derived xenografts without notable toxicity versus normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen while demonstrating a toolkit for applying these strategies more broadly.

Although incorporation of photo‐activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine‐modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross‐linking mass spectrometry (XL‐MS), we developed a diazirine‐modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α‐helical peptide LAVA20. We observed an unexpected backfolding of the diazirine‐containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL‐MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes., Can diazirine‐modified lipids be used to locate the position of UV‐induced cross‐links between this lipid and a membrane peptide by using MS? Although DiazPC fulfils all prerequisites for a photo‐activatable membrane lipid to study protein/lipid interactions and the cross‐links can be exactly assigned, we observed a backfolding of the lipid's alkyl chain. This is a general phenomenon for any type of membrane lipids with a hydrophilic group in the alkyl chain.

The high abundance of most viruses in infected host cells benefits their structural characterization; endogenous viruses are present in low copy numbers, however, and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity while stacked dsRNA bundles and the sub-stoichiometric polymerase localize underneath the capsid surface. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cellextract-based high-resolution structural virology.

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy’s bodies in neurons, which are the hallmark of Parkinson’s Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of α-syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between α-syn molecules in liquid droplets. In this work, we show that the conformational ensemble of α-syn shifts from a ‘hairpin-like’ structure towards more ‘elongated’ conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa3 oxidase supercomplex (bcc-aa3 supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa3 supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa3 supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa3 supercomplex physically associate.

Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.

Aneuploidy is a hallmark of cancer, but its complex nature limits our understanding of how it drives oncogenesis. Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia and is associated with markedly increased leukemia risk. Here, we propose that disequilibrium of the RUNX1 isoforms is key to the pathogenesis of trisomy 21 (i.e. Down syndrome)-associated myeloid leukemia (ML-DS). Hsa21-focused CRISPR-Cas9 screens uncovered a strong and specific RUNX1 dependency in ML-DS. Mechanistic studies revealed that excess of RUNX1A isoform – as seen in ML-DS patients – synergized with the pathognomonic Gata1s mutation in leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium or pharmacological interference with MYC:MAX dimerization. Our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidies, and opens new avenues for developing specific and targeted therapies.Graphical Abstract

The tumor suppressor protein p53 is a transcription factor that is referred to as the "guardian of the genome" and plays an important role in cancer development. p53 is active as a homotetramer; the S100β homodimer binds to the intrinsically disordered C-terminus of p53 affecting its transcriptional activity. The p53/S100β complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100β exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100β complexes by ESI-MS, XL-MS, and SPR. Wild-type p53 and single amino acid variants, representing different oligomeric states of p53 were individually investigated regarding their binding behavior towards S100β. The stoichiometry of the different p53/S100β complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100β dimer. In addition, XL-MS revealed the topologies of the p53/S100β complexes to be independent of p53's oligomeric state. With SPR, the thermodynamic parameters were determined for S100β binding to tetrameric, dimeric, or monomeric p53 variants. Our data prove that the S100β homodimer binds to different oligomeric states of p53 with similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100β interaction.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

Just recently, chemical cross-linking combined with mass spectrometry (XL-MS) has emerged as valuable tool to study protein interaction networks on the system-wide level. The current challenges in XL-MS are to develop robust workflows enabling a comprehensive capture of dynamic biological assemblies in their native environment in a routine manner. In this review, we will highlight both the latest technological developments as well as selected applications of XL-MS for investigating protein networks in cells, organisms, and tissue. In addition, different bioinformatics tools for data analysis will be presented. In light of these exciting new developments, XL-MS can be expected to become one of the most versatile techniques to address important biological questions in a timely manner.

The mineralocorticoid receptor (MR) with its ligand aldosterone (aldo) physiologically regulates electrolyte homeostasis and blood pressure but it can also lead to pathophysiological effects in the cardiovascular system. Previous results show that posttranslational modifications (PTM) can influence MR signaling and function. Based on in silico and in vitro data, casein kinase 1 (CK1) was predicted as a candidate for MR phosphorylation. To gain a deeper mechanistic insight into MR activation, we investigated the influence of CK1 on MR function in HEK cells. Co-immunoprecipitation experiments indicated that the MR is located in a protein-protein complex with CK1α and CK1ε. Reporter gene assays with pharmacological inhibitors and MR constructs demonstrated that especially CK1ε acts as a positive modulator of GRE activity via the C-terminal MR domains CDEF. CK1 enhanced the binding affinity of aldosterone to the MR, facilitated nuclear translocation and DNA interaction of the MR, and led to expression changes of pathophysiologically relevant genes like Per-1 and Phlda1. By peptide microarray and site-directed mutagenesis experiments, we identified the highly conserved T800 as a direct CK1 phosphorylation site of the MR, which modulates the nuclear import and genomic activity of the receptor. Direct phosphorylation of the MR was unable to fully account for all of the CK1 effects on MR signaling, suggesting additional phosphorylation of MR co-regulators. By LC/MS/MS, we identified the MR-associated proteins NOLC1 and TCOF1 as candidates for such CK1-regulated co-factors. Overall, we found that CK1 acts as a co-activator of MR GRE activity through direct and indirect phosphorylation, which accelerates cytosolic-nuclear trafficking, facilitates nuclear accumulation and DNA binding of the MR, and increases the expression of pathologically relevant MR-target genes.

Mass spectrometry (MS) has become one of the key technologies of structural biology. In this review, the contributions of chemical cross-linking combined with mass spectrometry (XL-MS) for studying three-dimensional structures of proteins and for investigating protein-protein interactions are outlined. We summarize the most important cross-linking reagents, software tools, and XL-MS workflows and highlight prominent examples for characterizing proteins, their assemblies, and interaction networks in vitro and in vivo. Computational modeling plays a crucial role in deriving 3D-structural information from XL-MS data. Integrating XL-MS with other techniques of structural biology, such as cryo-electron microscopy, has been successful in addressing biological questions that to date could not be answered. XL-MS is therefore expected to play an increasingly important role in structural biology in the future.

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.TOC Graphic

Graphical abstract We describe a rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 min per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/μL. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC-MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC-MS/MS methods in a routine clinical environment. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03614-y.

We describe a rapid liquid chromatography/mass spectrometry (LC/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 minutes per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/µl. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC/MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC/MS methods in a routine clinical environment.

Given the plasticity of hematopoietic stem/progenitor cells, multiple routes of differentiation must be blocked during acute myeloid leukemia pathogenesis – the molecular basis of which is incompletely understood. Here we report that post-transcriptional repression of transcription factor ARID3A by miR-125b is a key event in megakaryoblastic leukemia (AMKL) pathogenesis. AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing this disease. We show that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncover Arid3a as the main miR-125b target underlying this synergy. We demonstrate that during normal hematopoiesis this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. While Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a dual megakaryocytic/erythroid differentiation blockade and subsequently AMKL. Inversely, restoring ARID3A expression relieves the megakaryocytic differentiation arrest in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of post-transcriptional gene regulation interplay to block multiple routes of hematopoietic differentiation and cause leukemia. Surmounting this differentiation blockade in megakaryoblastic leukemia by restoring the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.Key pointsRepression of megakaryocytic transcription factor ARID3A by miR-125b synergizes with GATA1s to induce leukemiaRestoring ARID3A expression relieves megakaryocytic differentiation arrest in megakaryoblastic leukemiaGraphical abstract

Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein–protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2–3 days to complete. Valuable structural information can be derived by chemical cross-linking of proteins followed by mass spectrometry of the products. Iacobucci et al. use MS-cleavable reagents, enrichment by strong-cation-exchange chromatography, and LC-MS/MS analysis.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22151-x

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells., This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 in bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data reveal that the α-helical domain connecting the kinase homology and catalytic domains can acquire different conformations. Also, the XL-MS data of native ROS-GC1 in bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell., Chemical and photo‐crosslinkers genetically incorporated into β‐arrestins allow the mapping of footprints of different receptors on the arrestin surface in living cells, and to identify intermolecular pairs of proximal amino acids in GPCR‐arrestin complexes.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HG-SOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here we show, that the IGF2 mRNA binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HG-SOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in a RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signaling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rational for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HG-SOC.Graphical Abstract