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.

Staphylococcal Complement Evasion Protein Sbi Stabilises C3d Dimers by Inducing an N-Terminal Helix Swap.

Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.

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.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant.

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming tripartite complexes that act as competitive antagonists of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of the complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.