The L1624Q Variant in SCN1A Causes Familial Epilepsy Through a Mixed Gain and Loss of Channel Function

Variants of the SCN1A gene encoding the neuronal voltage-gated sodium channel NaV1.1 cause over 85% of all cases of Dravet syndrome, a severe and often pharmacoresistent epileptic encephalopathy with mostly infantile onset. But with the increased availability of genetic testing for patients with epilepsy, variants in SCN1A have now also been described in a range of other epilepsy phenotypes. The vast majority of these epilepsy-associated variants are de novo, and most are either nonsense variants that truncate the channel or missense variants that are presumed to cause loss of channel function. However, biophysical analysis has revealed a significant subset of missense mutations that result in increased excitability, further complicating approaches to precision pharmacotherapy for patients with SCN1A variants and epilepsy. We describe clinical and biophysical data of a familial SCN1A variant encoding the NaV1.1 L1624Q mutant. This substitution is located on the extracellular linker between S3 and S4 of Domain IV of NaV1.1 and is a rare case of a familial SCN1A variant causing an autosomal dominant frontal lobe epilepsy. We expressed wild-type (WT) and L1642Q channels in CHO cells. Using patch-clamp to characterize channel properties at several temperatures, we show that the L1624Q variant increases persistent current, accelerates fast inactivation onset and decreases current density. While SCN1A-associated epilepsy is typically considered a loss-of-function disease, our results put L1624Q into a growing set of mixed gain and loss-of-function variants in SCN1A responsible for epilepsy.

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.

Epistasis shapes the fitness landscape of an allosteric specificity switch

Epistasis is a major determinant in the emergence of novel protein function. In allosteric proteins, direct interactions between inducer-binding mutations propagate through the allosteric network, manifesting as epistasis at the level of biological function. Elucidating this relationship between local interactions and their global effects is essential to understanding evolution of allosteric proteins. We integrate computational design, structural and biophysical analysis to characterize the emergence of novel inducer specificity in an allosteric transcription factor. Adaptive landscapes of different inducers of the designed mutant show that a few strong epistatic interactions constrain the number of viable sequence pathways, revealing ridges in the fitness landscape leading to new specificity. The structure of the designed mutant shows that a striking change in inducer orientation still retains allosteric function. Comparing biophysical and functional properties suggests a nonlinear relationship between inducer binding affinity and allostery. Our results highlight the functional and evolutionary complexity of allosteric proteins.

Rational design and evaluation of the recombinant multiepitope protein for serodiagnosis of rubella

Background: Rubella is an infection caused by rubella virus (RV) and is generally regarded as a mild childhood disease. The disease continues to be of public health importance mainly because when the infection is acquired during early pregnancy it often results in fetal abnormalities, which are classified as congenital rubella syndrome (CRS). An accurate diagnosis for rubella is thus of pivotal importance for proper treatment. Objective: To produce a recombinant multiepitope protein (rMERUB) for the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2. Methods: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house ELISA assay. Results : Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to perform structural assays and an IgG ELISA. Structural analyses reveal rMERUB has a β leaf pattern that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples (22=positive; 11=negative) was performed using in-house ELISA and this was compared with a commercial kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99). Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays. Conclusions: The careful choice of epitopes and the high epitope density, coupled with simple-step purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the development of a diagnostic kit.

Implications of critical nodes-dependent unidirectional cross-talk between Plasmodium and Human SUMO

The endoparasitic pathogen, Plasmodium falciparum (Pf), modulates protein-protein interactions to employ post-translational modifications like SUMOylation in order to establish successful infections. The interaction between E1 and E2 (Ubc9) enzymes governs species specificity in the Plasmodium SUMOylation pathway. Here, we demonstrate that a unidirectional cross-species interaction exists between Pf-SUMO and Human-E2, whereas Hs-SUMO1 failed to interact with Pf-E2. Biochemical and biophysical analysis revealed that surface-accessible Aspartates of Pf-SUMO determine the efficacy and specificity of SUMO-Ubc9 interactions. Furthermore, we demonstrate that critical residues of the Pf-Ubc9 N-terminal are responsible for the lack of interaction between Hs-SUMO1 and Pf-Ubc9. Mutating these residues to corresponding Hs-Ubc9 residues restore electrostatic,pi-pi, and hydrophobic interactions and allows efficient cross-species interactions. We suggest that the critical changes acquired on the surfaces of Plasmodium SUMO and Ubc9 proteins as nodes can help Plasmodium exploit the host SUMOylation machinery. Thus, Pf-SUMO interactions can be targeted for developing antimalarials.

Strengthening Semaglutide-GLP-1R Binding Affinity Via a Val27-Arg28 Exchange in the Peptide Backbone of Semaglutide: A Computational Structural Approach

Semaglutide is a glucagon-like peptide 1 analog used for the treatment of patients with type 2 diabetes mellitus. With 94% sequence similarity to human GLP-1, semaglutide is a glucagon-like peptide-1 receptor (GLP-1R) agonist, which binds directly to GLP-1R, causing various beneficial downstream effects that reduce blood glucose. Incorporating currently (June 21, 2021) available experimental structural data in PDB of semaglutide and GLP-1R, and with a set of computational structural and biophysical analysis, this short paper for the first time puts forward an experimentally testable hypothesis: semaglutide is able to bind tighter to GLP-1R via a simple Val27-Arg28 exchange in its peptide backbone.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loop's C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts.