Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution.

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical GPCR that plays a central role in the inhibition of lipolytic and atherogenic activities. Its activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. GPR109A continues to be a target for developing potential therapeutics in dyslipidemia with minimized flushing response. Here, we present cryo-EM structures of the GPR109A in complex with dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures elucidate the binding mechanism of agonists, molecular basis of receptor activation, and insights into biased signaling elicited by some of the agonists. The structural framework also allows us to engineer receptor mutants that exhibit G-protein signaling bias, and therefore, our study may help in structure-guided drug discovery efforts targeting this receptor.

AlphaFold: Research accelerator and hypothesis generator.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

Cryo-EM research in India.

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

The expanding footprint of basic biology research in India.

Basic biology research in India has expanded in recent years, and addressing key challenges should enable this momentum to continue.

Molecular insights into atypical modes of ß-arrestin interaction with seven transmembrane receptors.

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Team science: building, nurturing, and expanding research collaborations.

Science is a collaborative endeavor, and the importance of collaborations across disciplines and boundaries is becoming clearer with the advent of new technologies. This article focuses on key aspects of initiating and sustaining new collaborations, and expanding from bilateral to multilateral efforts to create major impact through team science.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Editor Profile: Arun Shukla.

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal, and future directions in their field. Professor Arun Shukla is a Senior Fellow of DBT Wellcome Trust India Alliance, and Professor and Sonu Agrawal Memorial Chair at the Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India. He has served as an Editorial Board Member of The FEBS Journal since 2021.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

Ultrasound-assisted synthesis of MSNs/PS nanocomposite membranes for effective removal of Cd2+ and Pb2+ ions from aqueous solutions.

Contamination of heavy metal (Cd2+ & Pb2+) ions in drinking water is producing major impacts on the environment and public health and is considered one of the greatest dangers to humanity. Membrane technology has been chosen over other processing methods due to its simplicity and high capacity for more effective removal of hazardous heavy metals. In the current study, amine, thiol, and bi-thiol functional groups were used to functionalize mesoporous silica nanoparticles (MSNs) to improve the efficiency of the silica nanoparticle. The morphology of the MSNs as well as the existence of amine and thiol on the surface of MSNs was demonstrated by a variety of characterization techniques, including FTIR, TEM, and SEM examination. The impact of surface-modified MSNs on the morphology, properties, and performance of polysulfone (PS) nanofiltration (NF) membranes was also evaluated. The membrane that incorporated amine with thiol-based MSNs (DiMP-MSNs/PS-NF membrane) had the highest pure water permeability (6.7 LMH bar-1). As a result of the functional groups, the surface-modified MSNs/PS nanofiltration are extremely effective at removing heavy metal ions from aqueous solutions. The surface-modified MSNs/PS nano-filtration membranes exhibit unprecedented Cd2+ and Pb2+ removal rates of approximately 82% and 99%, respectively. This research indicates the possible application of the surface-modified MSNs/PS nanofiltration membrane as a promising platform to remove heavy metal ions from polluted water.

A key GPCR phosphorylation motif discovered in arrestin2⋠CCR5 phosphopeptide complexes.

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

Plasma membrane preassociation drives ß-arrestin coupling to receptors and activation.

ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

A streamlined protocol for expression and purification of wild-type ß-arrestins.

The two isoforms of ß-arrestins namely ß-arrestin 1 and 2 interact with, and regulate a broad repertoire of G protein-coupled receptors (GPCRs). While several protocols have been described in the literature for purification of ß-arrestins for biochemical and biophysical studies, some of these protocols involve multiple complicated steps that prolong the process and yield relatively smaller amounts of purified proteins. Here, we describe a simplified and streamlined protocol for expression and purification of ß-arrestins using E. coli as an expression host. This protocol is based on N-terminal fusion of GST tag and involves a two-step protocol involving GST-based affinity chromatography and size exclusion chromatography. The protocol described here yields sufficient amounts of high-quality purified ß-arrestins suitable for biochemical and structural studies.

Au-courant and novel technologies for efficient doubled haploid development in barley (Hordeum vulgare L.).

Bounteous modern and innovative biotechnological tools have resulted in progressive development in the barley breeding program. Doubled haploids developed (homozygous lines) in a single generation is significant. Since the first discovery of haploid plants in 1920 and, in particular, after discovering in vitro androgenesis in 1964 by Guha and Maheshwari, the doubled haploidy techniques have been progressively developed and constantly improved. It has shortened the cultivar development time and has been extensively used in: genetic studies, gene mapping, marker/trait association, and QTL studies. In barley, the haploid occurrence developed gradually from being a sporadic and random process (spontaneous) to haploid development by in vivo method of modified pollination or by in vitro culture of immature male or female gametophytes. Although significant improvement in DH induction protocols has been made, challenges still exist for improvement in areas such as: low efficiency, albinism, genotypic specificity etc. Here, the paper focuses on: haploidization via different in vitro, in vivo techniques, the recent advances technologies like centromere-mediated haploidization, hap induction gene, and Doubled haploid CRISPR. The au-courant work of different researchers in barley using these technologies is reviewed. Studies on different factors affecting haploid induction and work on genome doubling of barley haploids to produce DH lines via spontaneous and induced technologies has also been highlighted.

Structural snapshot of a ß-arrestin-biased receptor.

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to ß-arrestins (ßarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.