Structures of full-length glycoprotein hormone receptor signaling complexes

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are members of the glycoprotein hormone family essential to human reproduction and are important therapeutic drugs. They activate the same G protein-coupled receptor, LHCGR, by binding to the large extracellular domain (ECD). Here we report four cryo-EM structures of LHCGR, two wildtype receptor structures in the inactive and active states, and two constitutively active mutated receptor structures. The active structures are bound to CG and Gs heterotrimer, with one of the structure also containing the allosteric agonist, Org43553. The structures reveal a distinct ′push and pull′ mechanism of receptor activation, in which the ECD is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain (TMD). A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the ECD-TMD interface functions as a tethered agonist to induce conformational changes in TMD and G-protein coupling. Org43553 binds to a TMD pocket and interacts directly with P10 that further stabilizes the receptor in the active conformation. Together, these structures provide a common model for understanding glycoprotein hormone signal transduction and dysfunction, and inspire the search for clinically suitable small molecular compounds to treat endocrine diseases.

Structural Basis for the Diminished Ligand Binding and Catalytic Ability of Human Fetal-Specific CYP3A7

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the β3-β4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.

Characteristics of the Iron-responsive Element (IRE) Stems in the Untranslated Regions of Animal mRNAs

Background: Iron-responsive Elements (IREs) are hairpin structures located in the 5’ or 3’ untranslated region of some animal mRNAs. IREs have a highly conserved terminal loop and a UGC/C or C bulge five bases upstream of the terminal loop, which divides the hairpin stem into an upper stem and a lower stem. Objective: The objective of this study was to investigate the base-pair composition of the upper and lower stems of IREs to determine whether they are highly conserved among mRNAs from different genes. Methods: The mRNA sequences of six 5’IREs and five 3’IREs from several animal species were retrieved from the National Center for Biotechnology Information. The folding free energy of each IRE mRNA sequence was predicted using the RNAfold WebServer. Results: We found that the upper and lower stems of IREs are not highly conserved among the mRNAs of different genes. There are no statistically significant differences in the IRE structures or folding free energies between mammalian and non-mammalian species relative to either the ferritin heavy chain 5’IRE or ferroportin 5’IRE. There are no overall significant differences in the folding free energies between UGC/C-containing 5’IREs and C-bulge-containing 5’IREs, or between 5’IREs and 3’IREs. Conclusion: Further studies are needed to investigate whether the variations in IRE stem composition are responsible for fine-tuning the IRE/Iron-Regulatory Protein interactions among different mRNAs to maintain the balance of cellular iron metabolism, and to identify whether evolutionary processes drive the base-pair composition of the upper and lower stems of IREs toward any particular configuration.

The Reovirus μ2 C-Terminal Loop Inversely Regulates NTPase and Transcription Functions versus Binding to Factory-Forming μNS and Promotes Replication in Tumorigenic Cells

ABSTRACT The wild-type reovirus serotype 3 Dearing PL strain (T3wt) is being heavily evaluated as an oncolytic and immunotherapeutic treatment for cancers. Mutations that promote reovirus entry into tumor cells were previously reported to enhance oncolysis; here, we aimed to discover mutations that enhance the postentry steps of reovirus infection in tumor cells. Using directed evolution, we found that reovirus variant T3v10M1 exhibited enhanced replication relative to that of T3wt on a panel of cancer cells. T3v10M1 contains an alanine-to-valine substitution (A612V) in the core-associated μ2, which was previously found to have nucleoside-triphosphatase (NTPase) activities in virions and to facilitate virus factory formation by association with μNS. Paradoxically, the A612V mutation in μ2 from T3v10M1 was discovered to impair NTPase activities and RNA synthesis, leading to a 5-fold higher probability of abortive infection for T3v10M1 relative to that with T3wt. The A612V mutation resides in a previously uncharacterized C-terminal region that juxtaposes the template entry site of the polymerase μ2; our findings thus support an important role for this domain during virus transcription. Despite crippled onset of infection, T3v10M1 exhibited greater accumulation of viral proteins and progeny during replication, leading to increased overall virus burst size. Both far-Western blotting and coimmunoprecipitation (Co-IP) approaches corroborated that the A612V mutation in μ2 increased association with the nonstructural virus protein μNS and enhances burst size. Together, the data show that mutations in the C-terminal loop domain of μ2 inversely regulate NTPase and RNA synthesis versus interactions with μNS, but with a net gain of replication in tumorigenic cells. IMPORTANCE Reovirus is a model system for understanding virus replication and also a clinically relevant virus for cancer therapy. We identified the first mutation that increases reovirus infection in tumorigenic cells by enhancing postentry stages of reovirus replication. The mutation is in a previously uncharacterized C-terminal region of the M1-derived μ2 protein, which we demonstrated affects multiple functions of μ2, namely, NTPase, RNA synthesis, inhibition of antiviral immune response, and association with the virus replication factory-forming μNS protein. These findings promote a mechanistic understanding of viral protein functions. In the future, the benefits of μ2 mutations may be useful for enhancing reovirus potency in tumors.

The N-Terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition

<div> <div> <p>DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-specific changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step towards the efficient design of anti-virulence agents that target these proteins.</p> </div> </div>

RNA pull-down-Confocal Nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-Synuclein levels

ABSTRACTRNA-protein interactions are central to all gene expression processes and contribute to variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here we developed a fluorescent on-bead screening platform: RNA pull-down-Confocal Nanoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7’s primary target is an mRNA of α-Synuclein, which contributes to aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-Synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides novel methodology to search for RNA-protein interaction modulators.

The C-terminal loop of Arabidopsis thaliana guanosine deaminase is essential to catalysis

Guanosine deaminase (GSDA) in plants specifically deaminates (de)guanosine a nd deoxyguanosine to produce xanthosine with high specificity, which is further converted to xanthine, a key intermediate in purine metabolism and and uric...

Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3–5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.