Vibrational spectroscopy analysis of ligand efficacy in human M₂ muscarinic acetylcholine receptor (M₂R)

振動分光法を駆使した薬剤効能測定法の開発 --アセチルコリン受容体を標的とした神経疾患の治療薬開発への期待--. 京都大学プレスリリース. 2021-12-01.The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M₂ muscarinic acetylcholine receptor (M₂R)). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M₂R) and paves the path to rationally design ligands with varied efficacy towards the target GPCR

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex

熱、炎症などに関与するプロスタグランジン受容体EP3シグナリング複合体の可視化 --緑内障、高眼圧症治療薬の合理的設計に貢献--. 京都大学プレスリリース. 2022-09-15.Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E₂. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment-mediated crystallography

Fv antibody fragments have been used as co-crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large-scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram-positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site-specific protease during the purification process to yield tag-free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT-based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA-approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies

Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy

Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure (Kd) with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the (Kd) of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43

A Liquid Chromatography-Mass Spectrometry Method to Study the Interaction between Membrane Proteins and Low-Molecular-Weight Compound Mixtures

Molecular interaction analysis is an essential technique for the study of biomolecular functions and the development of new drugs. Most current methods generally require manipulation to immobilize or label molecules, and require advance identification of at least one of the two molecules in the reaction. In this study, we succeeded in detecting the interaction of low-molecular-weight (LMW) compounds with a membrane protein mixture derived from cultured cells expressing target membrane proteins by using the size exclusion chromatography-mass spectrometry (SEC-MS) method under the condition of 0.001% lauryl maltose neopentyl glycol as detergent and atmospheric pressure chemical ionization. This method allowed us to analyze the interaction of a mixture of medicinal herbal ingredients with a mixture of membrane proteins to identify the two interacting ingredients. As it does not require specialized equipment (e.g., a two-dimensional liquid chromatography system), this SEC-MS method enables the analysis of interactions between LMW compounds and relatively high-expressed membrane proteins without immobilization or derivatization of the molecules

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag

<div><p>There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQH<u>V</u>T-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.</p></div

Determination of minimum epitope sequence of Ra62 antibody.

<p>(A,B) Narrowing epitope region of Ra62 antibody by AlphaScreen. Biotin-SrtA-DRD1 C-terminal fragments were cell-free synthesized. Interactions between DRD1 fragments and Ra62 or Ra48 antibodies were evaluated by AlphaScreen (panel A) and Western blotting (panel B). Error bars in panel A indicate standard deviation (n = 4). Synthesis of biotinylated fusion protein was visualized by using anti-biotin antibody (panel B). (C) Ra62 antibody recognized 5 residues of DRD1 at its C-terminus. C-terminal DRD1 7-residue fragment was fused with Venus protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178246#pone.0178246.ref026" target="_blank">26</a>], and alanine mutation and deletion were introduced into it. Mutant proteins were synthesized by wheat cell-free system, and subjected to Western blotting with Ra62 antibody and anti-rabbit IgG-HRP as primary antibody. Synthesis of Venus protein was visualized by using anti-GFP antibody. (D) Ra62 antibody recognized terminus carboxyl group in the epitope peptide. Interaction between antibody and biotinylated peptide was detected using AlphaScreen. (E) Specificity of Ra62 antibody. Extracts from wheat germ or cultured cells were subjected to Western blotting using Ra62 antibody as primary antibody. Cell-free synthesized DRD1 was applied as positive control recognized by Ra62 antibody. CF, reaction mixture of cell-free synthesis containing wheat germ extract.</p

Purification of soluble proteins by CP5 system.

<p>(A) Purification of CYLD-CP5. Cell-free synthesized CYLD-CP5 was purified by CP5 system. Each fraction was subjected to SDS-PAGE and CBB staining. (B) Purification of control Venus-CP5 by CP5 system. (C) DUB assay. Linear tetra ubiquitin chain was treated with purified CYLD-CP5 and Venus-CP5. Degradation of products was visualized by SDS-PAGE and Ruby staining. (D) Purification of cell-free synthesized MARCH3-CP5 by CP5 system. Each fraction was subjected to SDS-PAGE and CBB staining. (E) Self-ubiquitination assay of purified MARCH3-CP5. Purified MARCH3-CP5 was mixed with E1, E2 (UbcH6), and HA-ubiquitin. Reaction mixture was applied to Western blotting. Ubiquitination was detected by HRP conjugated anti-HA antibody. (F) Recovery rate of soluble proteins purified by CP5 system. Signal intensity of each band in CBB staining images was measured by using ImageJ software. After background noise level was subtracted, signal intensity of CP5 tagged protein was plotted. Figures on the bar graph indicate recovery rate calculated by dividing recovered target proteins (elution 1 to 3) by total captured proteins (elution 1 to 3 and resin).</p