Biology subjects

Carlsson, J.

Publications and source records attributed to Carlsson, J..

17 recordsLinked to original sources

bioRxiv2026

Structural Dynamics of the Dopamine D2 Receptor with a Non-Basic Ligand

Progress in understanding protein-ligand interactions is revolutionizing drug design, especially for G protein-coupled receptors (GPCRs), which are targets for 35% of marketed drugs.1 The dopamine D2 receptor (D2R) represents a key drug target in schizophrenia and Parkinsons disease.2 While structural studies have clarified its interactions with classical ligands, the behavior of atypical, non-basic ligands like D2AAK2 remains unclear. Notably, D2AAK2 shows strong selectivity for D2R over the closely related D3R, despite identical binding pocket composition. Here, we present a cryo-EM structure of D2AAK2 bound to D2R, showing that aspartate 3.32 serves as the main anchoring point, even though the compound lacks a basic nitrogen atom. Using enhanced sampling molecular dynamics simulations and experimental approaches, we uncover a complex binding energy landscape. Simulations suggest that D2AAK2 receptor subtype selectivity between identical binding sites arises from different energy barriers for their conformational changes. Non-basic ligands offer advantages such as better brain penetration and improved pharmacokinetics.3,4 This study provides the first structural insights into a non-basic ligand targeting D2R, paving the way for developing more effective, selective drugs.

pharmacology and toxicology↗

bioRxiv2025

Structure-guided allosteric modulation of the delta opioid receptor

Opioid analgesics remain essential for pain management but are associated with significant adverse effects, including respiratory depression, tolerance, and dependence. The {delta}-opioid receptor ({delta}OR) represents a promising therapeutic target for developing safer opioid analgesics with reduced adverse effects compared to conventional -opioid receptor-targeting drugs. Positive allosteric modulators (PAMs) offer advantages over direct agonists by enhancing endogenous opioid signaling while preserving natural spatiotemporal activation patterns, potentially avoiding tolerance and dependence issues. Here, we present high-resolution cryo-EM structures of {delta}OR complexed with the peptide agonist DADLE and the PAM MIPS3614, revealing a novel lipid-facing allosteric binding site formed by transmembrane helices 2, 3, and 4. MIPS3614 stabilizes the active receptor conformation through a critical hydrogen bond with residue N1313.35 in the conserved sodium binding site, a key regulatory region controlling GPCR activation. Comprehensive mutagenesis, molecular dynamics simulations, and structure-activity relationships validate this proposed mechanism. Structure-guided optimization yielded MIPS3983 with enhanced binding affinity and retained cooperativity. Our findings establish the first molecular framework for {delta}OR allosteric modulation and provide a structural foundation for the rational design of safer opioid therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Revealing molecular determinants of ligand efficacy and affinity at the D2 dopamine receptor through molecular dynamics simulations

G protein-coupled receptors (GPCRs) control numerous physiological processes and are important therapeutic targets. Despite major research efforts, rational design of drugs that stimulate GPCR signaling is challenging because the molecular basis of activation remains poorly understood. Here, a combination of molecular dynamics simulations and pharmacological assays was used to study the activation mechanism of the D2 dopamine receptor (D2R), a major drug target for central nervous system diseases. Enhanced sampling simulations were performed to identify the key conformational changes involved in D2R activation by dopamine, and a computational platform for ligand profiling based on free energy calculations was developed. Simulations and experimental characterization of a series of dopamine derivatives showed that free energy calculations can predict the effect of small chemical modifications on ligand affinity and efficacy. Furthermore, simulations of D2 dopamine and {beta}2 adrenergic receptor activation revealed that ligand-induced activation of these GPCRs is driven by different molecular mechanisms despite recognizing chemically similar catecholamine agonists. Whereas dopamine interactions with the sixth transmembrane helix primarily drive activation of the D2R, hydrogen bonding with the fifth helix is the key interaction for activation of the {beta}2 adrenergic receptor. Our results highlight the complexity of GPCR activation and illustrate how molecular simulations can provide mechanistic insight and quantitative predictions of ligand activity, enabling structure-based drug design. Significance statementG protein-coupled receptors (GPCRs) on the cell surface recognize ligands such as hormones and neurotransmitters. The binding of the ligand to the receptor leads to the activation of intracellular signaling pathways, a communication system that controls essential physiological functions. GPCRs are therefore important therapeutic targets, and many drugs exert effects by modulating their activity. Here, we use molecular simulations to study the activation mechanism of a dopamine receptor, which is implicated in neurodegenerative and neuropsychiatric disorders. We elucidate the mechanism of receptor activation and develop strategies to predict ligand affinity and efficacy. We also identify differsences in the activation mechanisms of dopamine and adrenergic receptors. These findings provide novel insights into receptor activation and can accelerate the drug discovery process.

biophysics↗

bioRxiv2025

Computational design of constitutively active mutants of Dopamine D2 receptor inspired by ligand-independent activation mechanisms

G protein-coupled receptors (GPCRs) can signal in the absence of agonists through constitutive activity. This activity can be enhanced by mutations, resulting in receptors known as constitutively active mutants (CAMs). Such receptors can be implicated in various physiological and pathophysiological conditions, and also offer significant therapeutic potential. However, the molecular basis of their constitutive activity remains unknown. To investigate how CAMs affect receptor activation, we employed enhanced sampling simulations to study the dopamine D2 receptor (D2R), a key target in central nervous system therapies. Free energy landscape analyses revealed that CAMs promote a conformational shift favoring an active state similar to the agonist-bound receptor. To then identify novel CAMs, we developed a comprehensive strategy combining structural comparison, in-silico residue scanning, and free energy calculations, validated by luminescence-complementation-based assays. Applied to D2R, this approach uncovered a new single-point CAM, D2R-I481.46W, which was functionally validated. Further investigation revealed that this mutation activates allosteric communication pathways primarily involving transmembrane helix 5, particularly Ser1945.43, underscoring its role in transmitting activation signals to the intracellular domain. These findings deepen our understanding of constitutive GPCR activity and demonstrate the utility of this framework for identifying CAMs as ligand-independent models for structural, cellular, and physiological studies.

biophysics↗

bioRxiv2023

Rise of the Cybercrabs: how digital cloning in an integrated taxonomic framework can support deep-sea exploration

Taxonomy has been a labour-intensive field of expertise based on hours of manual work and lengthy comments that are mainly bound to books and publications on a two-dimensional world. But every species described is a three-dimensional organism that needs to be seen, manipulated to be fully understood in its native shape. Nowadays, digital technology allows us to transform everyone in an avatar or a digital clone with ease, but collections do not provide many type specimens in a digital format. Here we present a simple approach used to study a specific deep-sea crab Segonzacia mesatlantica and provide online digital taxonomy across four repository sites. This offers the possibility to describe, exchange digitally and analyse specimens in its full 3D, establish their taxonomy and share them widely on online databases as well as physically by additive manufacturing to duplicate them in collections and outreach activities. Using an integrated taxonomic approach that included the use of 3D type specimens and molecular barcoding we provide evidence that the genus Segonzacia may be more diverse than previously understood

zoology↗

bioRxiv2022

A baseline for the genetic stock identification of Atlantic herring, Clupea harengus, in ICES Divisions 6.a, 7.b-c

Atlantic herring in ICES Divisions 6.a, 7.b-c comprises at least three populations, distinguished by temporal and spatial differences in spawning, which have until recently been managed as two stocks defined by geographic delineators. Outside of spawning the populations form mixed aggregations, which are the subject of acoustic surveys. The inability to distinguish the populations has prevented the development of separate survey indices and separate stock assessments. A panel of 45 SNPs, derived from whole genome sequencing, were used to genotype 3,480 baseline spawning samples (2014-2021). A temporally stable baseline comprising 2,316 herring from populations known to inhabit Division 6.a was used to develop a genetic assignment method, with a self-assignment accuracy >90%. The long-term temporal stability of the assignment model was validated by assigning archive (2003-2004) baseline samples (270 individuals) with a high level of accuracy. Assignment of non-baseline samples (1,514 individuals) from Division 6.a, 7.b-c indicated previously unrecognised levels of mixing of populations outside of the spawning season. The genetic markers and assignment models presented constitute a toolbox that can be used for the assignment of herring caught in mixed survey and commercial catches in Division 6.a into their population of origin with a high level of accuracy.

zoology↗

bioRxiv2021

The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

Environmental DNA (eDNA) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the cytochrome oxidase I (COI) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA or to remove nontarget DNA, prior to amplification, by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan COI (mlCOIintF) and bacterial 16S (515F). Additionally, each step of the protocol was assessed by amplifying for both metazoan COI (mlCOIintF/jgHCO2198) and bacterial 16S (515F/806R) to investigate the effect on the metazoan and bacterial communities. Bacterial read abundance increased significantly in response to the captures (COI library), while the quality of the captured DNA was also improved. The metazoan-based probe captured bacterial DNA in a range that was also amplifiable with the 16S primers, demonstrating the ability of universal capture probes to isolate larger fragments of DNA from eDNA. Although the use of the tested COI probe cannot be recommended for metazoan enrichment, based on the experimental results, the concept of capturing longer fragments could be applied to metazoan metabarcoding. By using a truly conserved site without a high-level taxonomic resolution as a target for capture, it may be possible to isolate DNA fragments large enough to span over a nearby barcoding region (e.g., COI), which can then be processed through a conventional metabarcoding-by-amplification protocol.

molecular biology↗

bioRxiv2021

Comparative analysis of single nucleotide polymorphisms and microsatellite markers for parentage verification and discovery within the equine Thoroughbred breed

Short tandem repeat (STR), also known as microsatellite markers are currently used for genetic parentage verification within equine. Transitioning from STR to single nucleotide polymorphism (SNP) markers to perform equine parentage verification is now a potentially feasible prospect and a key area requiring evaluation is parentage testing accuracies when using SNP based methods, in comparison to STRs. To investigate, we utilised a targeted equine genotyping by sequencing (GBS) panel of 562 SNPs to SNP genotype 309 Thoroughbred horses - inclusive of 55 previously parentage verified offspring. Availability of STR profiles for all 309 horses, enabled comparison of parentage accuracies between SNP and STR panels. An average sample call rate of 97.2% was initially observed, and subsequent removal of underperforming SNPs realised a pruned final panel of 516 SNPs. Simulated trio and partial parentage scenarios were tested across 12-STR, 16-STR, 147-SNP and 516-SNP panels. False-positives (i.e. expected to fail parentage, but pass) ranged from 0% for 147-SNP and 516-SNP panels to 0.003% when using 12-STRs within trio parentage scenarios, and 0% for 516-SNPs to 1.6% for 12-STRs within partial parentage scenarios. Our study leverages targeted GBS methods to generate low-density equine SNP profiles and demonstrates the value of SNP based equine parentage analysis in comparison to STRs - particularly when performing partial parentage discovery.

genetics↗

bioRxiv2021

Bacteria are everywhere, even in your COI marker gene data!

The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in eDNA metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units or/and amplicon sequence variants are retrieved from such studies and referred to as "dark matter", and do not get a taxonomic assignment with a reference sequence. For a thorough investigation of this dark matter, we have developed the Dark mAtteR iNvestigator (DARN) software tool. A reference COI-oriented phylogenetic tree was built from 1,240 consensus sequences covering all the three domains of life, with more than 80% of those representing eukaryotic taxa. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 retrieved from the Midori reference 2 database. Similarly, sequences from 559 bacterial genera and 41 archaeal were retrieved from the BOLD database. DARN makes use of the phylogenetic tree to investigate and quantify pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms such as bacteria and archaea are also amplified in eDNA samples and we suggest bacterial COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and you may find it as a Docker at https://hub.docker.com/r/hariszaf/darn. Author summaryDARN is a software approach aiming to provide further insight in the COI amplicon data coming from environmental samples. Building a COI-oriented reference phylogeny tree is a challenging task especially considering the small number of microbial curated COI sequences deposited in reference databases; e.g ~4,000 bacterial and ~150 archaeal in BOLD. Apparently, as more and more such sequences are collated, the DARN approach improves. To provide a more interactive way of communicating both our approach and our results, we strongly suggest the reader to visit this Google Collab notebook where all steps are described step by step and also this GitHub page where our results are demonstrated. Our approach corroborates the known presence of microbial sequences in COI environmental sequencing samples and highlights the need for curated bacterial and archaeal COI sequences and their integration into reference databases (i.e. Midori, BOLD, etc). We argue that DARN will benefit researchers as a quality control tool for their sequenced samples in terms of distinguishing eukaryotic from non-eukaryotic OTUs/ASVs, but also in terms of understanding the unknown unknowns.

bioinformatics↗

bioRxiv2020

Identification of ligand-specific G-protein coupled receptor states and prediction of downstream efficacy via data-driven modeling

G protein-coupled receptors (GPCRs) shift between inactive states and active signaling states, to which intracellular binding partners can bind. Extracellular binding of ligands stabilizes different receptor states and modulates the intracellular response via a complex allosteric process, which is not completely understood. Despite the recent advances in structure determination and spectroscopy techniques, a comprehensive view of the ligand-protein interplay remains a challenge. We derived free energy landscapes describing activation of the {beta}2 adrenergic receptor ({beta}2AR) bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics (MD) simulations. The resulting free energy landscapes reveal clear shifts towards active-like states at the G protein binding site for receptors bound to partial and full agonists compared to antagonists and inverse agonists. Not only do the ligands control the population of states, they also modulate the conformational ensemble of the receptor by tuning allosteric protein microswitches. We find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cAMP signaling responses, highlighting the predictive power of our approach. Using dimensionality reduction techniques, we could further assess the similarity between the unique conformational states induced by different ligands. Two distant hotspots governing agonism on transmembrane helices 5 and 7, including the conserved NPxxY motif, formed the endpoints of an allosteric pathway between the binding sites. Our results demonstrate how molecular dynamics simulations can further provide insights into the mechanism of GPCR regulation by ligands, which may contribute to the design of drugs with specific efficacy profiles.

biophysics↗

bioRxiv2019

GPCRmd uncovers the dynamics of the 3D-GPCRome

G protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new 3D molecular structures of GPCRs (3D-GPCRome) during the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. While experimentally-resolved structures undoubtedly provide valuable snapshots of specific GPCR conformational states, they give only limited information on their flexibility and dynamics associated with function. Molecular dynamics (MD) simulations have become a widely established technique to explore the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations requires efficient storage resources and specialized software, hence limiting the dissemination of these data to specialists in the field. Here we present the GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyse and share GPCR MD data. GPCRmd originates from a community-driven effort to create the first open, interactive, and standardized database of GPCR MD simulations. We demonstrate the power of this resource by performing comparative analyses of multiple GPCR simulations on two mechanisms critical to receptor function: internal water networks and sodium ion interaction.

biophysics↗

bioRxiv2019

Environmental (e)DNA detection of the invasive pink salmon Oncorhynchus gorbuscha during the 2017 Norwegian invasion

The pink salmon Oncorhynchus gorbuscha was introduced from its native range in the Pacific to Northwest Russia several times since the 1950s. While this species has been regularly observed in rivers in Northern Norway since that time, there has been an upsurge in the numbers of odd-year O. gorbuscha individuals observed in rivers in southern Norway in recent years, and particularly in 2017. Although the wide-scale effects of this species presence are currently uncertain, there are concerns regarding potential competition between O. gorbuscha and native species - most notably the Atlantic salmon Salmo salar. Environmental (e)DNA is becoming a widely used tool to monitor rare and invasive species in aquatic environments. In the present pilot study, primers and a probe were developed to detect O. gorbuscha from eDNA samples taken from a Norwegian river system where the species was observed. Water samples were taken at both upstream and downstream locations of the Lysakerelva river during Autumn 2017 (to coincide with spawning) and during late Spring 2018. Autumn samples were positive for O. gorbuscha at both sampling locations, whereas Spring samples showed positive detection of this species in the upstream region of the river, when smolt should have left, or be in the process of leaving the river. These findings reveal that eDNA-based methods can be used detect the presence of O. gorbuscha during their spawning season. This suggests that odd-year populations have the potential to become established in the studied river system. We recommend that eDNA sampling is repeated to determine whether individuals of this odd-year population have survived at sea and return to spawn. Our assay specificity tests indicate that the tools developed in the present study can be used for detection of O. gorbuscha in both Norwegian and other European river systems where presence/absence data is required. We also suggest some modifications to our methodology that may improve upon the detection capabilities of O. gorbuscha using eDNA.

genetics↗

bioRxiv2019

Energy landscapes reveal agonist’s control of GPCR activation via microswitches

Agonist binding to G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signaling pathways. Al-though high resolution structures of different receptor states are available, atomistic details of the allosteric signalling across the membrane remain elusive. We calculated free energy landscapes of the {beta}2 adrenergic receptors activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which sheds new light on how microswitches govern the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of the agonist BI-167107 is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. The connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y-Y motif), which leads to a larger population of active-like states at the G protein binding site. This coupling is augmented by protonation of the strongly conserved Asp792.50. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected microswitches. Characterization of these can be used to understand how ligands stabilize distinct receptor states and contribute to development drugs with specific signaling properties. The developed simulation protocol is likely transferable to other class A GPCRs. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/627026v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1dfcfb5org.highwire.dtl.DTLVardef@bdaf81org.highwire.dtl.DTLVardef@14d74e6org.highwire.dtl.DTLVardef@35cea9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

The Arrival of Steppe and Iranian Related Ancestry in the Islands of the Western Mediterranean

A series of studies have documented how Steppe pastoralist-related ancestry reached central Europe by at least 2500 BCE, while Iranian farmer-related ancestry was present in Aegean Europe by at least 1900 BCE. However, the spread of these ancestries into the western Mediterranean where they have contributed to many populations living today remains poorly understood. We generated genome-wide ancient DNA from the Balearic Islands, Sicily, and Sardinia, increasing the number of individuals with reported data from these islands from 3 to 52. We obtained data from the oldest skeleton excavated from the Balearic islands (dating to [~]2400 BCE), and show that this individual had substantial Steppe pastoralist-derived ancestry; however, later Balearic individuals had less Steppe heritage reflecting geographic heterogeneity or immigration from groups with more European first farmer-related ancestry. In Sicily, Steppe pastoralist ancestry arrived by [~]2200 BCE and likely came at least in part from Spain as it was associated with Iberian-specific Y chromosomes. In Sicily, Iranian-related ancestry also arrived by the Middle Bronze Age, thus revealing that this ancestry type, which was ubiquitous in the Aegean by this time, also spread further west prior to the classical period of Greek expansion. In Sardinia, we find no evidence of either eastern ancestry type in the Nuragic Bronze Age, but show that Iranian-related ancestry arrived by at least [~]300 BCE and Steppe ancestry arrived by [~]300 CE, joined at that time or later by North African ancestry. These results falsify the view that the people of Sardinia are isolated descendants of Europes first farmers. Instead, our results show that the islands admixture history since the Bronze Age is as complex as that in many other parts of Europe.

genomics↗

bioRxiv2019

Field application of an eDNA assay for the threatened white-clawed crayfish Austropotamobius pallipes

The white-clawed crayfish Austropotamobius pallipes has undergone extensive declines within its native range in the last century. Because of its threatened status, European legislation requires the species to be regularly monitored and that Special Areas of Conservation (SACs) be designated for it. Knowledge on the distribution of this species is vital for addressing these needs. This study presents an environmental (e)DNA assay to detect A. pallipes in water samples, based on the mitochondrial cytochrome oxidase I (COI) gene, utilizing species-specific primers, a minor groove binding (MGB) probe and quantitative PCR. The results of this study indicate that eDNA is an effective tool for detecting A. pallipes in a lotic system, and could provide a valuable, non-invasive method for determining the distribution of this species.

genetics↗

bioRxiv2018

Mitochondrial D-loop DNA analyses of Norway Lobster (Nephrops norvegicus) reveals genetic isolation between Atlantic and Mediterranean populations

Nephrops norvegicus is a commercially valuable demersal fisheries species. Relatively little is understood about this species population dynamics across its distribution with previous mitochondrial and microsatellite studies failing to identify significant population-level differentiation. In this study, sequence variation in the mitochondrial (mtDNA) D-loop was analysed from samples across the distribution range. Analysis of a 375bp fragment of the D-loop revealed significant genetic differentiation between samples from the northeast Atlantic and the East Mediterranean (FST = 0.107, P<0.001). Tau ({tau}), theta ({theta}0 and {theta}1) and Fus Fs values suggest the species spread between 10,500 to 19,000 ybp and subsequently expanded rapidly across the Atlantic.

genetics↗

bioRxiv2017

A quantitative PCR based environmental DNA assay for detecting Atlantic salmon (Salmo salar L.)

O_LIThe Atlantic salmon (Salmo salar L.) has worldwide ecological, cultural and economic importance. The species has undergone extensive decline across its native range, yet concerns have been raised about its invasive potential in the Pacific. Knowledge on the distribution of this species is vital for addressing conservation goals.\nC_LIO_LIThis study presents an eDNA assay to detect S. salar in water samples, using quantitative PCR (qPCR) technology. Species-specific primers and a minor groove binding (MGB) probe were designed for the assay, based on the mitochondrial cytochrome oxidase I (COI) gene.\nC_LIO_LIThe results of this study indicate that eDNA is a highly sensitive tool for detecting S. salar in situ, and could potentially provide an alternative, non-invasive method for determining the distribution of this species.\nC_LI

genetics↗