Conformational flexibility within the small domain of human serine racemase

Serine racemase (SR) is a pyridoxal 5′-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,β-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78–155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded β-sheet of the small domain, including changes in the φ–ψ angles of all three residues in the C-terminal β-strand (residues 149–151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue β-strand (residues 78–83), but in the holoenzyme the β-strand is only four residues (78–81) and His82 has φ–ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target

Exploring Calbindin-IMPase fusion proteins structure and activity

Calbindin-D28k is a calcium binding protein that is highly expressed in the mammalian central nervous system. It has been reported that calbindin-D28k binds to and increases the activity of inositol Monophosphatase (IMPase). This is an enzyme that is involved in the homeostasis of the Inositol trisphosphate signalling cascade by catalysing the final dephosphorylation of inositol and has been implicated in the therapeutic mechanism of lithium treatment of bipolar disorder. Previously studies have shown that calbindin-D28k can increase IMPase activity by up to 250 hundred-fold. A preliminary in silico model was proposed for the interaction. Here, we aimed at exploring the shape and properties of the calbindin-IMPase complex to gain new insights on this biologically important interaction. We created several fusion constructs of calbindin-D28k and IMPase, connected by flexible amino acid linkers of different lengths and orientations to fuse the termini of the two proteins together. The resulting fusion proteins have activities 200%–400% higher the isolated wild-type IMPase. The constructs were characterized by small angle X-ray scattering to gain information on the overall shape of the complexes and validate the previous model. The fusion proteins form a V-shaped, elongated and less compact complex as compared to the model. Our results shed new light into this protein-protein interaction

The molecular basis for apolipoprotein E4 as the major risk factor for late onset Alzheimer's disease

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an -helical, 299-amino acid protein. Homozygosity for the ε4 allele is the major risk factor for developing late onset Alzheimer’s disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158 and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerisation state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small angle X-ray scattering, analytical ultracentrifugation, circular dichroism, X-ray fibre diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerise to form wavy filaments which do not share the characteristics of cross- amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis and these results open opportunities for uncovering new triggers for AD onset

Subtype selective y-Aminobutyric acid type A receptor (GABAAR) modulators acting at the benzodiazepine binding site: An update

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system (CNS) with fast, transsynaptic, and modulatory extrasynaptic effects being mediated by the ionotropic GABA type A receptors (GABAARs). These receptors are of particular interest because they are the molecular target of a number of pharmacological agents, of which the benzodiazepines (BZDs), such as diazepam, are the best described. The anxiolytic, sedating, and myorelaxant effects of BZDs are mediated by separate populations of GABAARs containing either α1, α2, α3, or α5 subunits and the molecular dissection of the pharmacology of BZDs indicates that subtype-selective GABAAR modulators will have novel pharmacological profiles. This is best exemplified by α2/α3-GABAAR positive allosteric modulators (PAMs) and α5-GABAAR negative allosteric modulators (NAMs), which were originally developed as nonsedating anxiolytics and cognition enhancers, respectively. This review aims to summarize the current state of the field of subtype-selective GABAAR modulators acting via the BZD binding site and their potential clinical indications

Crystallization and structure of ebselen bound to cysteine 141 of human inositol monophosphatase (IMPase)

Inositol monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47 Å resolution crystal structure (PDB entry 6zk0), are presented. In the complex with human IMPase, ebselen in a ring-opened conformation is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and in the crystal structure two adjacent dimers share four ebselen molecules, creating a tetramer with approximate 222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor or may block the binding of partner proteins

Co-crystallization of human inositol monophosphatase with the lithium mimetic L-690,330

Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit inositol monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by attenuating phosphatidylinositol (PI) cell signalling. Drug-discovery efforts have focused on small-molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L-690,330 is a potent bisphosphonate substrate-based inhibitor developed by Merck Sharp & Dohme. To aid future structure-based inhibitor design, determination of the exact binding mechanism of L-690,330 to IMPase was of interest. Here, the high-resolution X-ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 Å resolution is reported

Neurosteroid Modulation of Synaptic and Extrasynaptic GABAA Receptors of the Mouse Nucleus Accumbens

The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2βγ2) and extrasynaptic (α4βδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed

Pharmacological characterisation of MDI-222, a novel AMPA receptor positive allosteric modulator with an improved safety profile

There is considerable interest in positive allosteric modulators (PAMs) of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) subtype of ionotropic glutamate receptors as therapeutic agents for a range of cognitive and mood disorders. However, the challenge is to increase AMPA receptor (AMPAR) function sufficient to enhance cognitive function but not to the extent that there are mechanism-related pro-convulsant or convulsant side effects. In this present study, we report the preclinical pharmacology data for MDI-222, an AMPAR PAM which enhances cognition but has a much reduced side-effect (i.e. convulsant) liability relative to other molecules of this mechanism