Dissecting conformational rearrangements and allosteric modulation in metabotropic glutamate receptor activation

Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated to different brain diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on allosteric modulation of agonist action to be fully activated. Here we establish live cell compatible fluorescence labeling of mGlu2 by click chemistry through genetic code expansion. Using lanthanide resonance energy transfer, we establish multiple FRET sensors to monitor ligand effects on conformational changes in mGlu2 extracellular domain and subsequently dissect the underlying conformational states by smFRET. Using three distinct FRET sensors, we demonstrate that mGlu activation relies on a ligand-induced sampling of three conformational states. Orthosteric agonists act by promoting the closure of the mGlu2 ligand binding domains, leading to an equilibrium between an inactive intermediate and the active state. Allosteric modulator further push this equilibrium toward the active state, promoting and stabilizing the relative reorientation of the mGlu protomers. These results underline the complex and dynamic nature of such type of neuroreceptors, pointing out that ligands fine-tune activation by differentially acting on the equilibria between multiple states.

How Does the pH of Tree Bark Change with the Presence of the Epiphytic Bryophytes from the Family Orthotrichaceae in the Interaction with Trunk Inclination?

The pH of tree bark is affected by many factors, amongst them epiphytic bryophytes changing in their active state environment. Thus, we hypothesized that bryophytes can change bark acidity, dependently of the inclination of the branches, as inclination affect the water regime and particle deposition. We measured the pH under bryophyte cushions and compared it to nearby naked bark. Additionally, we compared results with experimental bark covering with neutral cover. We found that the pH of naked bark declines with decreasing inclination of trunks. Although bryophyte cover did not generally change the pH of the bark, there was a significant interaction with inclination: with higher inclination, bryophytes decrease the pH reaction of bark, while with lower inclination they increase it. One possible explanation may lie in changes to alkaline particle deposition, or conversely in the acidification of the bark by leaching. In addition, an experiment with a neutral cover showed that naked bark covering would substantially increase pH. As, on average, bryophytes do not change the pH of bark, there can be mutual interference between the alkalizing effect of the bark cover itself and the acidifying biological effect of bryophytes.

Structural insights into distinct signaling profiles of the μOR activated by diverse agonists

Drugs targeting the G protein-coupled μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression. While some partial opioid agonists appear to be safer than full agonists, the signaling pathways responsible for respiratory depression have yet to be elucidated. Here we investigated the structural and mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most potent and lethal opioids, and MP, a derivative from the kratom plant with reduced respiratory depression in animal studies at equianalgesic doses, exhibited markedly different signaling efficacy profiles for G protein subtype activation and recruitment of β-arrestins. Cryo-EM structures of the μOR-Gi1 complex with MP (2.5Å) and LFT (3.2Å) revealed that the two ligands engage distinct sub-pockets, and molecular dynamics (MD) simulations showed additional differences in the binding site that propagate to the intracellular side of the receptor where G proteins and β-arrestins bind. While MP favors the precise G protein-bound active state observed in the cryo-EM structures, LFT favors a distinct active state. These results highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes.

Creation of photocyclic vertebrate rhodopsin by single amino acid substitution

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photo-convert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, the molecular property of vertebrate rhodopsin successfully converted to the photocyclic and photoreversible properties by a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by the regulation of the recovery from the active state to the original dark state.

A molecular network of conserved factors keeps ribosomes dormant in the egg

Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally repressed. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules and occupy functionally important sites of the ribosome: a Habp4-eEF2 module that stabilizes ribosome levels and a Dap1b/Dapl1-eIF5a module that represses translation. Dap1b/Dapl1 is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Thus, a developmentally programmed, conserved ribosome state plays a key role in ribosome storage and translational repression in the egg.

Unravelling the Graded Millisecond Allosteric Activation Mechanism of Imidazole Glycerol Phosphate Synthase

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of key functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond timescale interconversion between these functional states hamper their detailed experimental and computational characterization. Here, we design a computational strategy tailored to reconstruct millisecond timescale events to describe the graded allosteric activation of imidazole glycerol phosphate synthase (IGPS) in the ternary complex. IGPS is a heterodimeric bienzyme complex responsible for the hydrolysis of glutamine to glutamate in the HisH subunit and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation pathway of wild-type IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that effector binding in HisF dramatically decreases the conformational barrier associated with the oxyanion hole formation in HisH, in line with the experimentally observed 4500-fold activity increase in glutamine production. The formation of the allosterically active state is controlled by time-evolving dynamic communication networks connecting the effector and substrate binding sites. This computational strategy can be generalized to study other unrelated enzymes undergoing millisecond timescale allosteric transitions.

Competitive Coherence Generates Qualia in Bacteria and Other Living Systems

The relevance of bacteria to subjective experiences or qualia is underappreciated. Here, I make four proposals. Firstly, living systems traverse sequences of active states that determine their behaviour; these states result from competitive coherence, which depends on connectivity-based competition between a Next process and a Now process, whereby elements in the active state at time n+1 are chosen between the elements in the active state at time n and those elements in the developing n+1 state. Secondly, bacteria should help us link the mental to the physical world given that bacteria were here first, are highly complex, influence animal behaviour and dominate the Earth. Thirdly, the operation of competitive coherence to generate active states in bacteria, brains and other living systems is inseparable from qualia. Fourthly, these qualia become particularly important to the generation of active states in the highest levels of living systems, namely, the ecosystem and planetary levels.

STUDY OF BLAZAR ACTIVITY IN 10 YEAR FERMI-LAT DATA AND IMPLICATIONS FOR TEV NEUTRINO EXPECTATIONS

Blazars are the most active extragalactic gamma-ray sources. They show sporadic bursts of activity, lasting from hours to months. In this work we present a 10-year analysis of a sample of bright sources detected by Fermi-LAT (100 MeV - 300 GeV). Using 2-week binned light curves (LC) we estimate the duty cycle (DC): fraction of time that the source spends in an active state. The objects show different DC values, with an average of 22.74% and 23.08% when considering (or not) the extragalactic background light ( EBL). Additionally, we study the so-called “blazar sequence” trend for the sample of selected blazars in the ten years of data. This analysis constrains a possible counterpart of sub-PeV neutrino emission during the quiescent states, leaving open the possibility to explain the observed IceCube signal during the flaring states.