Biology subjects

Gustavsson, M.

Publications and source records attributed to Gustavsson, M..

11 recordsLinked to original sources

bioRxiv2026

De novo macrocyclic peptide discovery produces potent class A GPCR ligands with diverse pharmacology

G protein-coupled receptors (GPCRs) represent one of the largest protein families of therapeutic importance, as over one third of all currently approved drugs target GPCRs. A growing proportion of GPCR drugs are peptides. However, class A GPCRs (over 80% of clinically studied targets) are underexploited as peptide targets, presumably since their only structured domain is the core, seven-helix, integral membrane fold presenting a limited solvent-exposed surface area. Screening libraries of de novo macrocyclic peptides have allowed for the targeting of otherwise intractable proteins, but their potential to generate selective ligands for class A GPCRs has not been demonstrated. Here, we used random nonstandard peptides integrated discovery (RaPID) to identify de novo macrocyclic peptide ligands targeting the chemokine receptor CXCR4. CXCR4 regulates cell migration, immune surveillance, and inflammation, making it an important therapeutic target for multiple diseases. We discovered novel, potent CXCR4-selective antagonists binding within the orthosteric pocket with nanomolar affinity. The most active peptides effectively inhibited receptor signaling, suppressed chemokine-driven cell migration, and blocked CXCR4-driven HIV entry. Our results show that even the structurally minimal class A GPCRs are viable targets for de novo cyclic peptide discovery, highlighting the potential for uncovering ligands towards other members of the GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2026

Assessing chemical toxicity across Eukaryota using multimodal transformers

Biodiversity is globally threatened by chemical pollution, yet toxicity data remain unavailable for millions of species and tens of thousands of chemicals, severely limiting our ability to assess ecological impacts. Here we present TRIDENT-2, a multimodal artificial intelligence model for predicting chemical toxicity across evolutionarily diverse eukaryotic species. Trained on 560,780 toxicity assays spanning 82,775 chemicals, 6,793 species, and multiple exposure scenarios, TRIDENT-2 accurately predicts toxicity across Eukaryota with an average median absolute error ranging from 1.76 to 3.80. By jointly learning from chemical, biological, and experimental information, it remains accurate across broad chemical and taxonomic distances, allowing for toxicity assessment for species and chemicals beyond the current experimental evidence. Our findings demonstrate that artificial intelligence can help overcome longstanding data limitations in ecotoxicology, paving the way for improved decision-making and reducing chemical impacts on biodiversity and ecosystems.

pharmacology and toxicology↗

bioRxiv2026

Chemokine receptor activity is differentially regulated by membrane cholesterol

Cholesterol is a key membrane component that regulates G protein-coupled receptor (GPCR) function, yet its molecular mechanisms remain unclear. Here, we combine chemical extraction of membrane sterols with functional signaling assays and single-molecule fluorescence resonance energy transfer (smFRET) to define how cholesterol controls activation of chemokine receptors. Reduction of membrane cholesterol in mammalian cells selectively decreased constitutive and agonist-induced signaling across CXCR1, CXCR2, CXCR4, while it activated ACKR3, and did not affect CXCR3, revealing receptor-specific dependence on membrane sterols. Mechanistically, cholesterol regulation partly required the conserved class A GPCR residue Trp4.50 and shifted agonist-bound CXCR4 toward active conformational states, providing a molecular explanation for its functional effects. In contrast, replenishment with oxidized cholesterol species failed to restore receptor activity, distinguishing cholesterol from oxysterols as modulators of receptor activation. Our findings identify cholesterol as an allosteric regulator of chemokine receptors and suggest that oxysterols may reshape inflammatory signaling by selectively modulating GPCR activity.

biochemistry↗

bioRxiv2026

Basal Internalization and Subcellular Localization of XCR1

Chemokines orchestrate immune cell trafficking through receptor-mediated signaling and are implicated in inflammatory, autoimmune, and neuropathic disorders. The XCL1-XCR1 axis is of particular interest because XCR1 is selectively expressed on mature conventional type 1 dendritic cells (cDC1s), where it supports communication with activated CD8+ T cells and NK cells and promotes antigen cross-presentation. This selectivity has made XCR1 an attractive target for dendritic cell-based cancer vaccines, while emerging evidence also links XCL1-XCR1 signaling to neuroinflammation and pain. Despite its therapeutic potential, the mechanisms governing XCR1 activation and trafficking remain understudied. Here, we characterize XCR1 expression, membrane trafficking, and basal internalization to define mechanisms that may influence therapeutic targeting. We show that XCR1 undergoes constitutive internalization through a {beta}-arrestin-independent but adaptor protein 2 (AP2)-dependent pathway, distinguishing it from other chemokine receptors with constitutive endocytosis. Furthermore, we identify specific sequence motifs critical for its subcellular localization and intracellular trafficking. These findings provide new mechanistic insights into XCR1 regulation and may inform the development of targeted therapeutics and antigen-delivery strategies in cancer and inflammation.

pharmacology and toxicology↗

bioRxiv2024

Integration of light quality signals regulates ABA abundance and stomatal movements during seedling establishment

Obtaining sufficient light for photosynthesis and avoiding desiccation are two key challenges faced by seedlings during early establishment. Perception of light quality via specialised photoreceptors signals the availability of sunlight for photosynthesis. Canopy shade is depleted in red (R) and enriched in far-red (FR) light, lowering R:FR ratio, while direct sunlight and sunflecks contain UV-B. The balance of these wavelengths can determine the developmental strategy adopted by seedlings to either avoid shade, via stem elongation, or promote the expansion of photosynthetic organs. How seedlings regulate stomatal movements in different light environments is poorly understood. Using FR and UV-B supplementation to mimic aspects of canopy shade and sunlight respectively, we monitored stomatal apertures in Arabidopsis thaliana cotyledons. We show that low R:FR inhibits stomatal opening via a mechanism involving PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and increased abscisic acid (ABA). In contrast, UV-B perceived by the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor antagonises this response to promote stomatal opening in a response requiring phototropin photoreceptors. The convergence of phytochrome and UVR8 signalling to control ABA abundance enables plants to coordinate stem elongation and water use during seedling establishment in dynamic light environments.

plant biology↗

bioRxiv2023

Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and {beta}-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.

biophysics↗

bioRxiv2023

ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding

Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.

biochemistry↗

bioRxiv2023

Transformers enable accurate prediction of acute and chronic chemical toxicity in aquatic organisms

Environmental safety assessments, as mandated by many regulations, require that toxicity data is generated for up to three trophic levels, algae, aquatic invertebrates, and fish. Conducting these tests in vivo is resource-intensive, time-consuming, and causes undue suffering. Computational methods are fast and cost-efficient alternatives, however, their adaptation in regulatory settings has been slow, both due to low accuracy and narrow applicability domains. Here we present a new method for predicting chemical toxicity based on molecular structure. The method is based on a transformer, capturing structural features associated with toxicity, followed by a deep neural network that predicts the corresponding effect concentrations. After training on data from tens of thousands of exposure experiments, the model shows high predictive performance for each of the three trophic levels. Compared to commonly used QSAR methods, the model has both a larger applicability domain and a considerably lower error. In addition, training the model on data that combines multiple types of effect concentrations further improves the performance. We conclude that transformer-based models have the potential to significantly advance computational predictions of chemical toxicity and make in silico approaches a more attractive alternative when compared to animal-based exposure experiments.

pharmacology and toxicology↗

bioRxiv2021

In silico analysis of the fast-growing thermophile Geobacillus sp. LC300 using a novel genome-scale metabolic model

Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, iGEL601, of Geobacillus sp. LC300, an important part of the metabolic engineering pipeline. The model contains 601 genes, 1240 reactions and 1305 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. Using flux sampling, the model shows high similarity to experimentally determined reaction fluxes with both glucose and xylose as sole carbon sources. Furthermore, the model predicts previously unidentified by-products, closing the gap in the carbon balance for both carbon sources. Finally, iGEL601 was used to suggest metabolic engineering strategies to maximise production of five industrially relevant compounds. The suggested strategies have previously been experimentally verified in other microorganisms, and predicted production rates are on par with or higher than those previously achieved experimentally. The results highlight the biotechnological potential of LC300 and the application of iGEL601 for use as a tool in the metabolic engineering workflow.

systems biology↗

bioRxiv2020

A Bitopic Miniprotein Regulates a Membrane-Embedded Enzyme via Topological Allostery

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to {beta}-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzymes function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLNs cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzymes active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

biophysics↗

bioRxiv2020

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide crosslinking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 {beta}1-strand, while also recapitulating earlier findings from NMR, modeling and crystallography of homologous receptors. A crosslinking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N-terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor-targeting small molecules and biologics with novel pharmacology.

biochemistry↗