Biology subjects

Gossert, A. D.

Publications and source records attributed to Gossert, A. D..

7 recordsLinked to original sources

bioRxiv2026

Neuronal calcium sensors emerge as novel adenylyl cyclase 8 regulators

Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal calcium sensor hippocalcin-like protein 1 (HPCAL1) as a direct interaction partner of AC8. Using biochemistry, structural proteomics, integrative modeling, cryo-EM and cell-based FRET approaches, we show that HPCAL1 associates with AC8 in a Ca2+-dependent manner. HPCAL1 shows weak positive modulation of AC8 activity in vitro, with sub micromolar affinity. HPCAL1 interacts with flexible regulatory regions of AC8, including the C1b domain and the N- and C-terminal regions. Interestingly, other members of the neuronal calcium sensor family can bind AC8 via the same sites. Together, our study reveals a previously unrecognized mode of Ca2+-dependent AC8 recognition by a group of neuronal calcium sensor proteins, which may be relevant to context-dependent regulation of cAMP signaling.

biochemistry↗

bioRxiv2025

GRKs phosphorylate GPCR C-terminal peptides in a hierarchical manner

Responses from G protein-coupled receptors (GPCRs) are downregulated in a precisely orchestrated process called desensitization. This process consists of two major steps: phosphorylation of the receptor by GPCR kinases (GRKs), predominantly on its C-terminus, and recruitment of arrestin, resulting in different signaling outcomes. We carried out an NMR-based study of the phosphorylation patterns generated by GRK1 and GRK2 on C-terminal peptides of selected receptors (rhodopsin for GRK1, and {beta}1- and {beta}2-adrenergic receptors (ARs) for GRK2). Our data reveal that the kinases are promiscuous with respect to the substrate peptide, but produce clearly defined phosphorylation patterns on each substrate. We found pronounced differences in the rates at which certain residues are phosphorylated, in particular in the PXPP motifs in rhodopsin and {beta}1AR. These results show, that GRKs produce well-defined phosphorylation patterns in absence of further modulators like the full receptor or G{beta}{gamma}, and that the time profile of the phosphorylation barcode seems to be largely encoded in the minimal pair of C-terminal peptide and GRK. The data further suggest that arrestin might encounter different phosphorylation barcodes over time, potentially inducing different responses at different time points in the desensitization process.

biochemistry↗

bioRxiv2025

Molecular interplay between ComEC domains leads to efficient DNA translocation during natural transformation

Naturally competent bacteria can take up and incorporate free DNA from their environment using complex machinery that is endogenously encoded. This process is called natural transformation and is a key mechanism in the spread of antibiotic resistance amongst bacteria, including many human pathogens. All competent bacteria require ComEC to transport the transforming DNA across the cytoplasmic membrane. In addition to the transmembrane competence domain predicted to form the DNA channel, most ComEC orthologues additionally contain an oligonucleotide binding (OB) domain and {beta}-lactamase-like domain. Here, we provide in-depth characterisation of the nuclease activity of the {beta}-lactamase-like domain and the DNA binding activity of the OB domain, and present high-resolution structures of both domains. We show that the in vitro nuclease activity of the {beta}-lactamase-like domain is enhanced when the OB domain is encoded on the same polypeptide chain. Additionally, we identify a pin loop within the {beta}-lactamase-like domain responsible for melting the DNA duplex prior to cleavage of the non-translocating strand, and a DNA channel lined with aromatic residues that guide the uncleaved translocating strand through ComEC. On the basis of our biochemical, structural and functional characterisation, we provide a mechanistic model for how ComEC achieves the simultaneous tasks of DNA degradation and translocation, central to the natural transformation process.

microbiology↗

bioRxiv2024

Human Cells for Human Proteins: Isotope Labeling in Mammalian Cells for Functional NMR Studies of Disease-Relevant Proteins

In biological and biomedical research the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods: it allows studying structure, function and dynamics of biomolecules and, importantly, their interactions with natural ligands or drugs. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in e.g. HEK293 cells are often established, isotope labeling is difficult and very expensive. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells. We demonstrate uniform 15N and 13C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV 13C-methyl labeling pattern. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. This enables NMR studies of important, but difficult to produce proteins, like receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.

biophysics↗

bioRxiv2023

Intrinsically disordered regions in the transcription factor MYC:MAX provide DNA selectivity via intramolecular interactions

The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large parts an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promotors containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins controls MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e. as intramolecular interactions. These findings are supported by Surface Plasmon Resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights how bHLH-LZ TFs are regulated by intramolecular interactions, which opens up new possibilities for drug discovery.

biophysics↗

bioRxiv2022

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/507991v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@183d480org.highwire.dtl.DTLVardef@1f4235aorg.highwire.dtl.DTLVardef@13d1aedorg.highwire.dtl.DTLVardef@b2c87f_HPS_FORMAT_FIGEXP M_FIG C_FIG The ongoing pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further drug development.

biophysics↗

bioRxiv2021

The Molecular Basis of FimT-mediated DNA Uptake during Bacterial Natural Transformation

Naturally competent bacteria encode sophisticated protein machineries for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. In this study, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We used NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimTs role in this process. FimT directly interacts with DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in FimTs conformationally flexible C-terminal tail. We also show that FimT orthologues from other {gamma}-Proteobacteria share the ability to bind to DNA. Our functional characterisation and comprehensive bioinformatic analysis of FimT, suggest that it plays an important role for DNA uptake in a wide range of competent species.

microbiology↗