Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b.

Sarafotoxins (SRTXs) are endothelin-like peptides extracted from snake venom. SRTXs stimulate the endothelin ETA and ETB receptors and enhance vasoconstriction, followed by left ventricular dysfunction and bronchoconstriction. SRTXs include four major isopeptides, S6a-d, with different subtype selectivities. Here, we report the crystal structure of the human ETB receptor in complex with the non-selective sarafotoxin S6b at 3.0 Å resolution. This structure reveals the similarities and differences between the binding modes of the endothelins and S6b. Moreover, molecular dynamics simulations based on the S6b-bound receptor provides structural insight into the subtype selectivity of the sarafotoxins. Our study clarifies the recognition mechanism of the endothelin-like peptide families.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation.

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation.

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.