CD46 is a complement regulator with important immune-related roles. CD46 functions as a pathogen receptor and is a potent co-stimulator for the induction of interferon-γ (IFN-γ)-secreting T helper 1 (TH1) effector T cells and their subsequent switch into interleukin-10 (IL-10)-producing regulatory T cells. Here, we identify the Notch protein family member Jagged1 as a new physiological ligand for CD46. Further, CD46 regulates Notch receptors and ligands expression during T cell activation and disturbance of the CD46-Notch crosstalk impedes IFN-γ induction and IL-10 switching. Importantly, CD4+ T cells from CD46-deficient patients and patients with hypomorphic Jagged1 mutations (Alagille Syndrome) fail to mount appropriate TH1 responses in vitro and in vivo suggesting that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.

The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.glycosylation | mass spectrometry | X-ray

Highlights► We review the high resolution structures of the Notch receptor and ligands. ► Highlight the docking events of Notch receptor and ligand at the cell surface. ► Indicate the future challenges in understanding Notch receptor–ligand interactions.

Abstract2 H spin relaxation NMR experiments to study the dynamics of deuterated backbone α-positions, D α , are developed. To date, solution-state 2 H relaxation measurements in proteins have been confined to side-chain deuterons -primarily 13 CH 2 D or 13 CHD 2 methyl groups. It is shown that quantification of 2 H relaxation rates at D α backbone positions and the derivation of associated order parameters of C α -D α bond vector motions in small [U-15 N, 13 C, 2 H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins -ubiquitin (8.5 kDa) at 10°C, 27°C, and 40°C, and a variant of GB1 (6.5 kDa) at 22°C. In both proteins, the D α -derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from 15 N relaxation rates. Semi-quantitative solution state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for D α sites in proteins equal to 174 kHz. Using the uniform value of QCC for all D α sites, we show that C α -D α bond vectors are motionally distinct from the backbone amide N-H bond vectors, with 2 H-derived squared order parameters of C α -D α bond vector motions, S 2 CαDα , on average slightly higher than their N-H amides counterparts, S 2 NH . For ubiquitin, the 2 H-derived backbone mobility compares well with that found in a 1-μs molecular dynamics simulation.

No abstract

SummaryThe Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.

Type 4 filaments (T4F)—of which type 4 pili (T4P) are the archetype—are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis . We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis . Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F.