Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Pump-like channelrhodopsins: Not just bridging the gap between ion pumps and ion channels.

Channelrhodopsins are microbial rhodopsins that work as light-gated ion channels. Their importance has become increasingly recognized due to their ability to control the membrane potential of specific cells in a light-dependent manner. This technology, termed optogenetics, has revolutionized neuroscience, and numerous channelrhodopsin variants have been isolated or engineered to expand the utility of optogenetics. Pump-like channelrhodopsins (PLCRs), one of the recently discovered channelrhodopsin subfamilies, have attracted broad attention due to their high sequence similarity to ion-pumping rhodopsins and their distinct properties, such as high light sensitivity and ion selectivity. In this review, we summarize the current understanding of the structure-function relationships of PLCRs and discuss the challenges and opportunities of channelrhodopsin research.

Endogenous ligand recognition and structural transition of a human PTH receptor.

Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins.

Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

Structure-Function Relationship of Channelrhodopsins.

Ion-translocating rhodopsins, especially channelrhodopsins (ChRs), have attracted broad attention as a powerful tool to modulate the membrane potential of cells with light (optogenetics). Because of recent biophysical, spectroscopic, and computational studies, including the structural determination of cation and anion ChRs, our understanding of the molecular mechanism underlying light-gated ion conduction has been greatly advanced. In this chapter, I first describe the background of rhodopsin family proteins including ChR, and how the optogenetics technology has been established from the discovery of first ChR in 2002. I later introduce the recent findings of the structure-function relationship of ChR by comparing the crystal structures of cation and anion ChRs. I further discuss the future goal in the fields of ChR research and optogenetic tool development.

Author Correction: Structural insights into µ-opioid receptor activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of the neurotensin receptor 1 in complex with ß-arrestin 1.

Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human ß-arrestin 1 (ßarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with ßarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.

Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding.

G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states.

Cortical layer-specific critical dynamics triggering perception.

Perceptual experiences may arise from neuronal activity patterns in mammalian neocortex. We probed mouse neocortex during visual discrimination using a red-shifted channelrhodopsin (ChRmine, discovered through structure-guided genome mining) alongside multiplexed multiphoton-holography (MultiSLM), achieving control of individually specified neurons spanning large cortical volumes with millisecond precision. Stimulating a critical number of stimulus-orientation-selective neurons drove widespread recruitment of functionally related neurons, a process enhanced by (but not requiring) orientation-discrimination task learning. Optogenetic targeting of orientation-selective ensembles elicited correct behavioral discrimination. Cortical layer-specific dynamics were apparent, as emergent neuronal activity asymmetrically propagated from layer 2/3 to layer 5, and smaller layer 5 ensembles were as effective as larger layer 2/3 ensembles in eliciting orientation discrimination behavior. Population dynamics emerging after optogenetic stimulation both correctly predicted behavior and resembled natural internal representations of visual stimuli at cellular resolution over volumes of cortex.

Conformational transitions of a neurotensin receptor 1-Gi1 complex.

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex.

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

Crystal structure of the natural anion-conducting channelrhodopsin GtACR1.

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Structural mechanisms of selectivity and gating in anion channelrhodopsins.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist.

The protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) involved in hemostasis, thrombosis, and inflammation, is activated by thrombin or other coagulation proteases. This activation is inhibited by the irreversible antagonist vorapaxar used for anti-platelet therapy. Despite detailed structural and functional information, how vorapaxar binding alters the structural properties of PAR1 to prevent activation is hardly known. Here we apply dynamic single-molecule force spectroscopy to characterize how vorapaxar binding changes the mechanical, kinetic, and energetic properties of human PAR1 under physiologically relevant conditions. We detect structural segments stabilizing PAR1 and quantify their properties in the unliganded and the vorapaxar-bound state. In the presence of vorapaxar, most structural segments increase conformational variability, lifetime, and free energy, and reduce mechanical rigidity. These changes highlight a general trend in how GPCRs are affected by strong antagonists.

Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6.

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

The light-driven sodium ion pump: A new player in rhodopsin research.

Rhodopsins are one of the most studied photoreceptor protein families, and ion-translocating rhodopsins, both pumps and channels, have recently attracted broad attention because of the development of optogenetics. Recently, a new functional class of ion-pumping rhodopsins, an outward Na+ pump, was discovered, and following structural and functional studies enable us to compare three functionally different ion-pumping rhodopsins: outward proton pump, inward Cl- pump, and outward Na+ pump. Here, we review the current knowledge on structure-function relationships in these three light-driven pumps, mainly focusing on Na+ pumps. A structural and functional comparison reveals both unique and conserved features of these ion pumps, and enhances our understanding about how the structurally similar microbial rhodopsins acquired such diverse functions. We also discuss some unresolved questions and future perspectives in research of ion-pumping rhodopsins, including optogenetics application and engineering of novel rhodopsins.

Role of Asn112 in a Light-Driven Sodium Ion-Pumping Rhodopsin.

Light-driven outward sodium-pumping rhodopsin (NaR) was recently found in marine bacteria. Krokinobacter eikastus rhodopsin 2 (KR2) actively transports sodium and lithium ions in NaCl and LiCl, respectively, while it pumps protons in KCl. NaR has a conserved NDQ (N112, D116, and Q123 in KR2) motif, and previous studies suggested an important role for N112 in the function of KR2. Here we replaced N112 with 19 different amino acids and studied the molecular properties of the mutants. All mutants exhibited absorption bands from a protonated Schiff base in the λmax range from 508 to 531 nm upon heterologous expression in Escherichia coli, whose ion-pumping activity was measured using pH electrodes. The function of these mutants was classified into three phenotypes: wild-type (WT)-like Na+/H+ compatible pump, exclusive H+ pump, and no pump. Among the 19 mutants, only N112D, -G, -S, and -T showed light-driven Na+ pump activity, N112A, -C, -P, -V, -E, -Q, -I, -L, -M, -F, and -W were exclusively H+ pumps, and N112H, -K, -Y, and -R exhibited no pump activity. The mutants of the no pump function lack a blue-shifted M intermediate, indicating that Schiff base deprotonation is a prerequisite for Na+ and H+ pumps. In contrast, the subsequent red-shifted O intermediate was observed for WT and N112V but absent for N112T and N112A, suggesting that observation of this intermediate depends on kinetics. Although N112D, -G, -S, and -T are able to pump Na+, they also pump H+ in NaCl, where Na+ and H+ pumps compete with each other because of the decreased Na+ uptake efficiency. From these facts, an exclusive Na+ pump in NaCl exists only in WT. We conclude that N112 is one of the functional determinants of NaR.