Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors.

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the ß-carboline, methyl 6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an 'upper' and 'lower' moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure-activity relationships, and empower future structure-based drug design campaigns.

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation.

Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor's glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.

Mechanisms of inhibition and activation of extrasynaptic αß GABAA receptors.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.

Novel Functional Properties of Missense Mutations in the Glycine Receptor ß Subunit in Startle Disease.

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and ß subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR ß subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR ß variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR ß subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and ß subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR ß subunits to gephyrin aggregates. However, a significantly lower number of GlyR ß aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR ß subunit aggregates was increased in comparison to wild-type ß. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR ß-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR ß M3-M4 loop, we suggest that structural changes within the GlyR ß subunit result in differences in GlyR ß-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Artemisinins and their impact on inhibitory neurotransmission.

Artemisinin, a major extract of the annual mugwort Artemisia annua, and its semisynthetic derivatives represent state-of-the-art antimalarial drugs. These compounds also target, via poorly understood mechanisms, various mammalian pathways, thereby exhibiting anticancer and immunomodulatory properties. Recently, crystal structures of artemisinins with two mammalian targets were determined, namely, gephyrin, the prime scaffolding protein at inhibitory postsynapses, and pyridoxal kinase, a central metabolic enzyme synthesizing vitamin B6. These structures and corresponding functional studies demonstrate that artemisinins play a dual role in modulating inhibitory synapses, acting on postsynaptic sites by impeding inhibitory neurotransmitter receptor clustering and on presynaptic terminals by limiting the biosynthesis of the inhibitory neurotransmitter γ-aminobutyric acid. These studies pave the way for further investigations of artemisinins as inhibitory neurotransmission modulators in humans.

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission.

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Šresolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia.

Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.

Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin.

γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3-M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the ß3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.

Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins.

The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Šresolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.

Structural Framework for Metal Incorporation during Molybdenum Cofactor Biosynthesis.

The molybdenum cofactor (Moco) is essential for the catalytic activity of all molybdenum-containing enzymes with the exception of nitrogenase. Moco biosynthesis follows an evolutionarily highly conserved pathway and genetic deficiencies in the corresponding human enzymes result in Moco deficiency, which manifests itself in severe neurological symptoms and death in childhood. In humans the final steps of Moco biosynthesis are catalyzed by gephyrin, specifically the penultimate adenylation of molybdopterin (MPT) by its N-terminal G domain (GephG) and the final metal incorporation by its C-terminal E domain (GephE). To better understand the poorly defined molecular framework of this final step, we determined high-resolution crystal structures of GephE in the apo state and in complex with ADP, AMP, and molybdate. Our data provide novel insights into the catalytic steps leading to final Moco maturation, namely deadenylation as well as molybdate binding and insertion.

Design and synthesis of high-affinity dimeric inhibitors targeting the interactions between gephyrin and inhibitory neurotransmitter receptors.

Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8 nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin-receptor interplay.

Molecular basis of the alternative recruitment of GABA(A) versus glycine receptors through gephyrin.

γ-Aminobutyric acid type A and glycine receptors (GABA(A)Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA(A)Rs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin-GABA(A)R α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR ß subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABA(A)R α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin-receptor interactions identify two residues as primary determinants for gephyrin's subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABA(A)Rs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin-GABA(A)R interaction.

Modulation of gephyrin-glycine receptor affinity by multivalency.

Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR ß-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR ß-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.

Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.