The domestic chicken exhibits extraordinarily high plumage diversity in sharp contrast to the uniform plumage phenotype in its wild ancestor, the red junglefowl. The melanocortin-1 receptor (MC1R) locus is the most variable pigmentation locus in domestic chicken. Here we have analyzed whole-genome sequence data from 10,026 birds, including red junglefowls and 393 populations of domestic chicken. We document ultrarapid evolution of 18 MC1R alleles, due to the accumulation of 9 missense mutations combined with reshuffling of sequence variants within the single MC1R exon. The lack of linkage disequilibrium at the MC1R locus strongly suggests that this combinatorial allele diversity is generated by interallelic gene conversion, possibly promoted by its high guanine-cytosine content. Functional analyses of MC1R variants expressed in transfected cells, using second-messenger and bioluminescence resonance energy transfer assays together with homology modeling, demonstrate that most missense mutations have significant effects on MC1R signaling and include both activating and inhibitory changes. One common MC1R allele differs by as many as three functionally relevant missense mutations from the most closely related wild-type allele, and its phenotype reflects their combined effects. Genome-wide analyses further show that MC1R acts as a modifier of dominant white and recessive white phenotypes caused by mutations in premelanosome protein and tyrosinase, respectively. In both cases, MC1R alleles promote a pure white phenotype, either by enhancing eumelanin pigmentation (dominant white) or inhibiting eumelanin pigmentation (recessive white).

DC-SIGN is a C-type lectin receptor expressed on antigen-presenting cells that is crucial for pathogen recognition and immune modulation. Here, we identify and characterize a previously unrecognized cryptic allosteric pocket in DC-SIGN using molecular dynamics simulations, NMR spectroscopy, cryogenic electron microscopy, and biochemical assays. Rotation of the gatekeeper residue M270 exposes the pocket whose occupancy modulates glycan binding. Mutations M270F and T314A mimic the occupied and unoccupied states of this pocket, respectively, shifting the conformational equilibrium of α-helix 2 and altering the oligosaccharide affinity via the extended carbohydrate binding site. While Ca2+ coordination at the canonical binding site remains unaffected, our data reveal a complex interplay between the Ca2+ binding sites and the canonical and extended glycan binding surfaces. These findings uncover a hierarchical allosteric mechanism that enables selective tuning of glycan affinity and suggest the cryptic pocket as a novel target for drug discovery in C-type lectins.

Metabolite-sensing G protein-coupled receptors (GPCRs), such as hydroxycarboxylic acid receptor 2 (HCA2), translate endogenous and microbial signals into physiological responses, regulating metabolism and immunity, yet the extent of HCA2 functional diversification across mammals remain unclear. Here, comparative pharmacology, metabolomics, evolutionary analysis, and structural mapping of mammalian HCA2 orthologs reveal extensive functional diversification across mammals, especially in odd-toed ungulates. Notably, African rhinoceros HCA2 exhibits multiple HCA3-like substitutions, resulting in loss of responsiveness to HCA2 agonists and increased sensitivity to HCA3-specific ligands. Positive selection analyses and metabolomic profiling of fecal extracts implicate microbiome-derived metabolites, particularly phenylpropionic and trans-cinnamic acid, as potential drivers of this adaptive shift. Mutagenesis experiments identified key amino acid substitutions in extracellular and transmembrane regions that modulate ligand potency and efficacy. These findings demonstrate that mammalian HCA2 receptors have undergone lineage-specific molecular evolution shaped by host-microbe metabolic interactions, highlighting how ecological contexts drive receptor adaptation and functional diversification.

Phytochromes are sensory photoreceptors in eukaryotes and prokaryotes that control physiological processes. In prototypical phytochromes, photoisomerization of the methine-bridged tetrapyrrole of the Pr state is the first step in (de)activating the photoreceptor. The underlying reaction sequence runs through a series of intermediate states. Among them, the Meta-Rc state plays a critical role since it precedes the formation of the Pfr state, which is linked to the functional secondary structure transition of the tongue, a phytochrome-specific peptide segment. In this work, we have studied the structure and reactions of Meta-Rc of the bacterial phytochrome Agp1 (Agrobacterium fabrum) by IR difference and resonance Raman spectroscopy. It is shown that the formation of Meta-Rc is associated with the enolization of the terminal ring D and the deprotonation of ring B or C, whereas reprotonation of the chromophore occurs with the decay of Meta-Rc. Proton migration represents the essential trigger for the secondary structure transition of the tongue since the β-sheet and α-helix structures can be interconverted by changing the pH. The pH-dependent conformational equilibrium is observed in Meta-Rc at 250 K and in Pfr at 290 K, albeit with different pKA values. The results show that the secondary structure transition is induced by chromophore-linked proton transfer steps rather than by conformational relaxations of the chromophore itself. In view of previous findings on the proton dependence of the reverse process in bathy phytochromes, we conclude that intramolecular proton transfer is an indispensable prerequisite for the secondary structure transition in phytochromes in general.

In this study, we present a strategy to uncouple tumor necrosis factor (TNF)-like cell death induction from TNFR2 agonism in a tumor-targeted fashion. Single-domain antibodies (sdAbs) targeting TNFR1 were generated by combining camelid immunization with yeast surface display. Reformatting of resulting paratopes as bispecific antibodies (bsAbs) in a 2 + 2 manner by employing an sdAb-based paratope targeting HER2 revealed the identification of an immunocytokine-like bsAb, referred to as immunocytokine mimetic (ICM), which triggered TNF-like tumor cell death of HER2-overexpressing cancer cells as well as robust caspase-1,-3, and-8 activation in a cis-targeted manner. By modulating the valency of the TNFR1-directed sdAb, killing capacities as well as caspase activities of HER2-targeted ICMs were significantly augmented, eventually resulting in enhanced cell death induction when compared with TNF. Moreover, HER2-targeted TNFR1 ICMs also displayed a beneficial, i.e., substantially reduced profile in inducing unconditional pro-inflammatory cytokine release from peripheral blood mononuclear cells (PBMCs).

Adhesion G protein-coupled receptors (aGPCRs) constitute a structurally and functionally distinct group within the superfamily of GPCRs. In 2015, the International Union of Pharmacology invited the Adhesion GPCR Consortium to publish a comprehensive review about aGPCRs and establish a unified nomenclature. Since then, substantial progress has been made in delineating the biological roles, molecular architecture, biochemical properties, expression profiles, ligand repertoire, and activation and signaling strategies of aGPCRs. Commensurate with these advances, their relevance to human pathophysiology has become increasingly apparent. In a coordinated effort, the Adhesion GPCR Consortium has reviewed recent progress in this field and provides a comprehensive assessment of the current understanding of aGPCR biology, including a focus on human and mammalian aGPCRs, their evolutionary origins, methodological approaches, and model systems for their investigation, as well as emerging approaches for their therapeutic targeting. SIGNIFICANCE STATEMENT: Adhesion G protein-coupled receptors are versatile cell-surface proteins that integrate structural, biochemical, and physiological functions, with major roles in health and disease. This review summarizes current knowledge of their molecular features, functions in diverse model systems, and emerging opportunities for therapeutic targeting, providing a comprehensive resource that connects basic biology with translational applications across multiple scientific disciplines.

Human trichromatic color vision relies on three cone opsins [long-, middle-, and short-wavelength-sensitive opsins (LWS-, MWS-, and SWS-opsins, respectively)], whereas scotopic rod vision is mediated by rhodopsin. Although the structure of rhodopsin was solved more than 20 years ago, cone opsin structures have been lacking. Here, we present cryo-electron microscopy structures of the three human cone opsins, each bound to a G protein and all-trans retinal in the presumed active state. All three cone opsins differ markedly from rhodopsin. Within the retinal binding pocket, we identified a distinct counterion site (LWS- and MWS-opsins) and a ring of serines around the retinal (SWS-opsin). The active cone opsin structures explain how amino acid substitutions fine-tune spectral sensitivity and help clarify the molecular basis of color vision deficiencies and key differences in rod versus cone activation.

The melanocortin-4 receptor is a G protein-coupled receptor and a key regulator of appetite and metabolism. It can interact with the melanocortin-receptor accessory protein 2, a single transmembrane helix protein known to interact with several different G protein-coupled receptors. However, the consequences of this interaction are not completely understood. Here we report that co-expression of melanocortin-receptor accessory protein 2 has multiple effects on the melanocortin-4 receptor: it enhances G protein-mediated signaling and simultaneously impairs β-arrestin2 recruitment and, consequently, internalization. In addition, co-expression of melanocortin-receptor accessory protein 2 leads to an increased number of monomers of melanocortin-4 receptor by disrupting receptor oligomers. A structural homology model of the active state melanocortin-4 receptor - melanocortin-receptor accessory protein 2 - Gαs complex suggests interaction sites that are relevant for receptor activation. Our data indicate that melanocortin-receptor accessory protein 2 is an accessory protein that interacts with and influences melanocortin-4 receptor structure, biasing its signaling towards G protein-mediated effects.

In phytochromes, photoisomerization of the chromophore and subsequent structural relaxations lead to the functionally essential secondary structure transition of the tongue, a phytochrome-specific protein segment. The coupling mechanism between chromophore and protein structural changes is yet not understood, but electric field changes are discussed to play an important role. In this work, electric field changes in the chromophore binding pocket (CBP) are confirmed to propagate over long distances through the protein and alter the electric field in the tongue region. An experimental-theoretical approach to analyze local electric fields using Stark reporters has been further developed. These are nitrile groups introduced site-specifically into the protein via noncanonical amino acids. The functional integrity of the variants is checked by crystallography and various spectroscopies. For the first time, functionally intact variants with substitutions in the tongue are generated. Based on frequency shifts and relative intensities of the nitrile stretching modes, hydrogen-bonding and noncovalent electric field contributions are separated. The field changes originating in the CBP are transduced to the tongue along a pathway via Phe192. Given a proper direction of the net electric field vector in the tongue region, the magnitude of the field may be sufficient to destabilize the tongue structure.

The 5th International Symposium on Regulatory Autoantibodies Targeting GPCR (RAB-GPCRs) advanced the understanding of the significant role played by autoantibodies targeting G-protein-coupled receptors (GPCRs) in various human diseases. Once considered passive markers, RAB-GPCRs are now recognized as active modulators of cellular signaling, immune regulation, and inflammation. The symposium highlighted their involvement in multiple prominent pathologies, including autoimmune diseases, cardio- and cerebrovascular diseases, and neuroimmunologic disorders such as myalgic encephalomyelitis/chronic fatigue syndrome and post-COVID-19 syndrome (ME/CFS/PCS), as well as solid organ and hematopoietic stem cell transplantation (SOT/HSCT). Experts from rheumatology, immunology, and neurology presented interdisciplinary discussions on the potential of RAB-GPCRs as biomarkers and therapeutic targets. Advances in screening methods, biomarker identification, and therapeutic strategies were shared, emphasizing their diagnostic potential and application in novel therapeutic interventions. This report summarizes key insights from the symposium, particularly focusing on the modulatory properties of RAB-GPCRs and their relevance in both immune-mediated diseases and other pathologies (e.g., vascular, degenerative) that are traditionally not considered primarily immune-mediated. Ongoing research is expected to further establish these autoantibodies as crucial components in disease modulation and systems biology contexts, offering new opportunities for precision medicine and improved clinical outcomes in immune-related disorders.

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

Neuronal morphogenesis is guided by filopodia, dynamically generated plasma membrane protrusions filled with parallel actin filaments. However, how filopodial actin filaments are locally produced, organized, and maintained remains unclear. The transmembrane protein PLPPR3 induces filopodia in neurons and other cells. We find that the intracellular domain (ICD) of PLPPR3 forms liquid condensates, which exhibit strong co-partitioning of actin monomers. These condensates promote actin polymerization within the condensates at the expense of actin monomers in the environment, consistent with thermodynamic coupling of actin partitioning and polymerization, which we recapitulate in a modified polymerization kinetics model. This mechanism requires favorable actin partitioning into the condensate relative to the environment. Using crosslinking mass spectrometry, we identify a WH2-like actin-affinity domain within the PLPPR3 ICD. Deleting this domain lowers actin partitioning in vitro and decreases filopodia formation in vivo . Our findings establish a previously unrecognized mechanism for actin network remodeling, in which condensates act as actin sinks, locally boosting monomer concentrations and facilitating polymerization of actin filaments. One sentence summary Our study uncovers PLPPR3 condensates to locally enrich actin monomers and promote actin polymerization, driving actin network remodeling and neuronal filopodia formation. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, SFB 958, SFB 1423, EXC 2008 European Molecular Biology Organization, ALTF 625-2022

The function of the biological photoswitch phytochrome is initiated by photoisomerization of the methine-bridged tetrapyrrole chromophore, followed by thermal relaxation steps. As a result of this reaction cascade, the protein interconverts between two parental state. These states, denoted as Pr (red absorbing) and Pfr (far-red absorbing), represent the physiologically inactive and active form of the protein, respectively. In this work we studied the primary photoprocesses of two bacterial phytochromes Agp1 and Agp2, in which either Pr or Pfr is the stable dark state, respectively. We employed cryogenic IR difference and resonance Raman spectroscopy between 4 K and 130 K to trap and characterize the species formed on the reaction pathways from Pfr to Lumi-F in Agp2 and Pr to Lumi-R in Agp1. The spectra analysis primarily focuses on the C[double bond, length as m-dash]O stretching modes, which are assigned based on isotopic labelling experiments. In both proteins, three sub-states were identified, which reveal similar patterns of sequential structural changes. In the first sub-state L1 of both photoreceptors, generated at 4 K, structural changes are restricted to the isomerization site including rings D and C. In L2, formed at 30 K in Agp2 but at the same temperature range with L1 in Agp1, the structural changes propagate to ring B, and in L3 also include ring A. Comparison with previously published studies demonstrates that the present approach of cryogenic vibrational spectroscopy provides important structural insights that complement results from crystallography and ultrafast time-resolved spectroscopy.

Disclosure: L. Ruck: None. P. Scheerer: None. S. Paisdzior: None. S. Wiegand: None. G. Kleinau: None. N. Heyder: None. P. Gmach: None. S. Jyrch: None. A. Beck-Sickinger: None. M. Troll: None. H. Biebermann: None. P. Kühnen: None. Introduction: The melanocortin 4 receptor (MC4R), a G protein-coupled receptor (GPCR), is a critical regulator of body weight within the hypothalamus and is embedded in the leptin-melanocortin signaling pathway. Heterozygous MC4R gene mutations have been identified as potent genetic risk factors for the development of obesity. Additionally, the MC4R agonist setmelanotide has recently been approved as a pharmacological treatment option for patients with certain rare monogenic forms of obesity. In recent years it has been described that MC4R related differential (biased) signaling is playing an important role for body weight regulation and the downstream effect of MC4R ligands. However, the interplay between MC4R genetic variants and different endogenous and external MC4R ligands remains elusive. Methods: We analyzed in vitro the signaling of 20 heterozygous MC4R mutations, which have been identified in a cohort of children with obesity, in regards to Gs, Gq/11, ERK, G12/13 and b-arrestin2 recruitment and after stimulation with different MC4R ligands (α-MSH, β-MSH, setmelanotide) in HEK293 cells. Additionally, MC4R mutations were further characterized by analysis of our previously solved the cryo-electron microscopic (cyro-EM) structures. Results: We observed a ligand and genetic variant dependent differential (bias) signaling of the MC4R. The “protective” MC4R variant V103I was associated with an increase of Gq signaling after stimulation with α-MSH. Contrary to the complete loss of function variants like Y80C, D90N and S127L (deficit in all analyzed pathways), MC4R variants as S77L or T178M led to a reduction of non-Gs signaling cascades while Gs signaling was not altered. These signaling profiles were ligand dependent, which was partially related to MC4R conformation changes, which were analyzed based on cryo-EM structure data. Conclusion: Our findings emphasize the critical role of differential (bias) signaling for MC4R function. Cryo-EMStructural data- combined with in vitro functional data allowed to gain further insights into the structural regulation of the MC4R, which can be relevant to optimized MC4R agonists as a treatment option for patients with certain forms of obesity. Presentation: Monday, July 14, 2025

Toll-like receptor 8 (TLR8) in humans senses RNA degradation products and elicits an inflammatory immune response. In contrast, the ligand specificity and function of its murine counterpart mTLR8, long considered non-functional, remain poorly defined. Here, we established an agonist combination model of poly-deoxythymidine (poly-dT) DNA and TLR7/8 binding site 1 agonists such as uridine or the benzazepine compound TL8-506, which activates mTLR8, while suppressing mTLR7 signaling. Extensive agonist analysis based on this model revealed that 2’,3’-cyclic guanosine monophosphate (2’,3’-cGMP) serves as a natural ligand for mTLR8, suggesting functionality of its binding site 1 without engagement of site 2. In addition, 2’,3’-cyclic uridine monophosphate, bacterial single-stranded (ss) DNA, double-stranded (ds) DNA fragments, microRNAs, ssRNA derived from HIV1, SARS-CoV-2, or bacterial sources all potentiate mTLR8 sensing of site 1 agonists. All these stimuli induce distinct inflammatory responses from murine macrophages and microglia via TLR8. In vivo , intrathecal administration of TL8-506 and poly-dT led to microglial accumulation and neuronal injury in the murine cerebral cortex through TLR8, highlighting the potential neuropathological consequences of mTLR8 activation. Taken together, our study defines mTLR8 as a nucleic acid sensor detecting 2’,3’-cGMP as well as combinations of ssDNA, dsDNA, ssRNA fragments, 2’,3’-cyclic nucleotide monophosphates, and nucleosides, with implications for host defense and neuroinflammation. ### Competing Interest Statement The authors have declared no competing interest.

Phytochromes are biliprotein photoreceptors found in bacteria, fungi, and plants. The soil bacterium Agrobacterium fabrum has two phytochromes, Agp1 and Agp2, which work together to control DNA transfer to plants and bacterial conjugation. Both phytochromes interact as homodimeric proteins. For fluorescence resonance energy transfer (FRET) measurements, various Agp1 mutants and wild-type Agp2 were labeled with specific fluorophores to study their interaction. FRET efficiencies rose from position 122 to 545 of Agp1. The photosensory chromophore module (PCM) of Agp1 did not show a FRET signal, but the PCM of Agp2 did. Docking models suggest that Agp1 and Agp2 interact with their histidine kinase and PCM perpendicular to each, around 45 amino acids of Agp1 or Agp2 are involved.

The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validated the RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the process of functional association between G proteins and GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit. In atomic detail, we reveal the temporal progression of structural rearrangements of GDP-bound heterotrimeric Gs protein (GsGDP) upon coupling to the β2-adrenergic receptor (β2AR) using molecular dynamics simulations. The binding of GsGDP to the β2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release, the rate-limiting step during G-protein activation. In particular, the opening of α1-αF helices, displacement of the αG helix, and the opening of the α-helical domain weaken the interaction between GDP and the G protein. Signal transduction to the G protein occurs via a novel receptor interface, confirmed by site-directed mutagenesis and functional assays. From this β2AR-GsGDP intermediate, the G protein must undergo an in-plane rotation along the receptor axis to reach the β2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface interact to transmit the signal over 30Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G protein coupling specificity.