Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.

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Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.

Neoplastic transformation often results in cells with unusual glycosylation patterns specific to the type and stage of different cancers, providing numerous venues for therapeutic and diagnostic reagents (1, 2). Lectins are non-catalytic proteins with strict specificity for individual mono-or oligosaccharides, which can make them powerful tools for detecting changes in the carbohydrate structure in glycoproteins and glycolipids.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological ‘intermediate’ conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Ryanodine receptors (RyRs) are intracellular Ca 2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.

Background: S25-26 displays remarkable specificity and avidity toward the unusual inner core LPS from Chlamydia. Results: Liganded and unliganded structures reveal bound antigen and significant ordered N-glycosylation on the variable heavy chain. Conclusion: Groove-type paratope recognizes the antigen in a manner distinct from all other Chlamydia-specific antibodies. Significance: Structural analysis of germ-line-coded antibodies provides insight to anaphylaxis induced by ␣-galactose (␣Gal) epitopes on therapeutic antibodies.

BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is often a life-threatening arrhythmia disorder with variable penetrance and expressivity. Little is known about the incidence or outcomes of CPVT patients with ≥2 variants.MethodsThe phenotypes, genotypes and outcomes of patients in the Pediatric and Congenital Electrophysiology Society CPVT Registry with ≥2 variants in genes linked to CPVT were ascertained. The American College of Medical Genetics & Genomics (ACMG) criteria and structural mapping were used to predict the pathogenicity of variants (3D model of pig RyR2 in open-state).ResultsAmong 237 CPVT subjects, 193 (81%) had genetic testing. Fifteen patients (8%) with a median age of 9 years (IQR 5–12) had ≥2 variants. Sudden cardiac arrest occurred in 11 children (73%), although none died during a median follow-up of 4.3 years (IQR 2.5–6.1). Thirteen patients (80%) had at least two RYR2 variants, while the remaining two patients had RYR2 variants plus variants in other CPVT-linked genes. Among all variants identified, re-classification of the commercial laboratory interpretation using ACMG criteria led to the upgrade from variant of unknown significance (VUS) to pathogenic/likely pathogenic (P/LP) for 5 variants, and downgrade from P/LP to VUS for 6 variants. For RYR2 variants, 3D mapping using the RyR2 model suggested that 2 VUS by ACMG criteria were P/LP, while 2 variants were downgraded to likely benign.ConclusionsThis severely affected cohort demonstrates that a minority of CPVT cases are related to ≥2 variants, which may have implications on family-based genetic counselling. While multi-variant CPVT patients were at high-risk for sudden cardiac arrest, there are insufficient data to conclude that this genetic phenomenon has prognostic implications at present. Further research is needed to determine the significance and generalizability of this observation. This study also shows that a rigorous approach to variant re-classification using the ACMG criteria and 3D mapping is important in reaching an accurate diagnosis, especially in the multi-variant population.