Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT₇) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT₇ with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. ¹⁵N NMR relaxation and H/D exchange experiments revealed that binding of dT₇ increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4.     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1^−/− growth plate cartilage but not in ChGn-2^−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1^−/−, and ChGn-2^−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.     

Change of RNase P RNA function by single base mutation correlates with perturbation of metal ion binding in P4 as determined by NMR spectroscopy

The solution structures of two 27 nt RNA hairpins and their complexes with cobalt(III)-hexammine [Co(NH₃)₆³⁺] were determined by NMR spectroscopy. The RNA hairpins are variants of the P4 region from Escherichia coli RNase P RNA: a U-to-A mutant changing the identity of the bulged nucleotide, and a U-to-C, C-to-U double mutant changing only the bulge position. Structures calculated from NMR constraints show that the RNA hairpins adopt different conformations. In the U-to-C, C-to-U double mutant, the conserved bulged uridine in the P4 wild-type stem is found to be shifted in the 3′-direction by one nucleotide when compared with the wild-type structure. Co(NH₃)₆³⁺ is used as a spectroscopic probe for Mg(H₂O)₆²⁺ binding sites because both complexes have octahedral symmetry and have similar radii. Intermolecular NOE crosspeaks between Co(NH₃)₆³⁺ and RNA protons were used to locate the site of Co(NH₃)₆³⁺ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop in both mutants, but is shifted 3′ by about one base pair in the double mutant. The change of the metal ion binding site is compared with results obtained on corresponding mutant RNase P RNA molecules as reported by Harris and co-workers (RNA, 1, 210–218).     

Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA^Phe forms a trinucleotide loop in solution, but Mg²⁺ and dimethylallyl modification of A₃₇ N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ₃₂, ψ₃₉, and A₃₇ C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ₃₂ on the anticodon stem-loop from E.coli tRNA^Phe. The ψ₃₂ modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ₃₂-A₃₈ and U₃₃–A₃₇ and the base of ψ₃₂ stacks between U₃₃ and A₃₁. The glycosidic bond of ψ₃₂ is in the anti configuration and is paired with A₃₈ in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ₃₂ modification increases the melting temperature of the stem by ~3.5°C, although the ψ₃₂ and U₃₃ imino resonances are exchange broadened. The results suggest that ψ₃₂ functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.     

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/Principal Findings

We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγ^null) mice engrafted with human hematopoietic stem cells. Human CD45⁺ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγ^null mice with HLA-A2⁺ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353_(111–119), NS4b 2423_(181–189), and NS4a 2148_(56–64).

Conclusions/Significance

This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.     

Interaction of C5 protein with RNA aptamers selected by SELEX

RNA aptamers binding to C5 protein, the protein component of Escherichia coli RNase P, were selected and characterized as an initial step in elucidating the mechanism of action of C5 protein as an RNA-binding protein. Sequence analyses of the RNA aptamers suggest that C5 protein binds various RNA molecules with dissociation constants comparable to that of M1 RNA, the RNA component of RNase P. The dominant sequence, W2, was chosen for further study. Interactions between W2 and C5 protein were independent of Mg²⁺, in contrast to the Mg²⁺ dependency of M1 RNA–C5 protein interactions. The affinity of W2 for C5 protein increased with increasing concentration of monovalent NH₄⁺, suggesting interactions via hydrophobic attraction. W2 forms a fairly stable complex with C5 protein, although the stability of this complex is lower than that of the complex of M1 RNA with C5 protein. The core RNA motif essential for interaction with C5 protein was identified as a stem–loop structure, comprising a 5 bp stem and a 20 nt loop. Our results strongly imply that C5 protein is an interacting partner protein of some cellular RNA species apart from M1 RNA.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.     

A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels

Auxiliary Ca²⁺ channel β subunits (Ca_Vβ) regulate cellular Ca²⁺ signaling by trafficking pore-forming α₁ subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3–GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca_Vβ SH3–GK module regulates multiple Ca²⁺ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca_Vβ SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3–GK interaction, and fully reconstitutes Ca_Vβ effects on channel trafficking, activation gating, and increased open probability (P_o). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3–GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3–GK interaction. Remarkably, this pair discriminated between Ca_Vβ trafficking and gating properties: α_1C targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P_o functions were selectively lost. A more extreme case, in which the trans SH3–GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the α_1C (Ca_V1.2) subunit. The results reveal that Ca_Vβ SH3 and GK domains function codependently to tune Ca²⁺ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca²⁺ channel activity.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β₂–α₂ loop region. This structure might provide new insights into the early events of conformational transition of PrP^C into PrP^Sc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β₂–α₂ loop and α₃ helix.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.