Molecular Determinant Underlying Selective Coupling of Primary G-Protein by Class A GPCRs.

G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.

Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

BBOF1 is required for sperm motility and male fertility by stabilizing the flagellar axoneme in mice.

The sperm flagellum is a specialized type of motile cilium composed of a typical "9 + 2" axonemal structure with peri-axonemal structures, such as outer dense fibers (ODFs). This flagellar arrangement is crucial for sperm movement and fertilization. However, the association of axonemal integrity with ODFs remains poorly understood. Here, we demonstrate that mouse BBOF1 could interact with both MNS1, an axonemal component, and ODF2, an ODF protein, and is required for sperm flagellar axoneme maintenance and male fertility. BBOF1 is expressed exclusively in male germ cells from the pachytene stage onwards and is detected in sperm axoneme fraction. Spermatozoa derived from Bbof1-knockout mice exhibit a normal morphology, however, reduced motility due to the absence of certain microtubule doublets, resulting in the failure to fertilize mature oocytes. Furthermore, BBOF1 is found to interact with ODF2 and MNS1 and is also required for their stability. Our findings in mice suggest that Bbof1 could also be essential for human sperm motility and male fertility, thus is a novel potential candidate gene for asthenozoospermia diagnosis.

Zygotic Vsx1 Plays a Key Role in Defining V2a Interneuron Sub-Lineage by Directly Repressing tal1 Transcription in Zebrafish.

In the spinal cord, excitatory V2a and inhibitory V2b interneurons are produced together by the final division of common P2 progenitors. During V2a and V2b diversification, Tal1 is necessary and sufficient to promote V2b differentiation and Vsx2 suppresses the expression of motor neuron genes to consolidate V2a interneuron identity. The expression program of Tal1 is triggered by a Foxn4-driven regulatory network in the common P2 progenitors. Why the expression of Tal1 is inhibited in V2a interneurons at the onset of V2a and V2b sub-lineage diversification remains unclear. Since transcription repressor Vsx1 is expressed in the P2 progenitors and newborn V2a cells in zebrafish, we investigated the role of Vsx1 in V2a fate specification during V2a and V2b interneuron diversification in this species by loss and gain-of-function experiments. In vsx1 knockdown embryos or knockout Go chimeric embryos, tal1 was ectopically expressed in the presumptive V2a cells, while the generation of V2a interneurons was significantly suppressed. By contrast, in vsx1 overexpression embryos, normal expression of tal1 in the presumptive V2b cells was suppressed, while the generation of V2a interneuron was expanded. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly bind to tal1 promoter and repress tal1 transcription. These results indicate that Vsx1 can directly repress tal1 transcription and plays an essential role in defining V2a interneuron sub-lineage during V2a and V2b sub-lineage diversification in zebrafish.

A Long Polymorphic GT Microsatellite within a Gene Promoter Mediates Non-Imprinted Allele-Specific DNA Methylation of a CpG Island in a Goldfish Inter-Strain Hybrid.

It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.