Sialoglycan binding triggers spike opening in a human coronavirus.

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.

Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway.

Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme.

Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron-transfer high-energy collision dissociation (EThcD), on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure.

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the "V" is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal ß-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and ß-propeller combination (Olfm1(coil-Olf)) that reveals a disulfide-linked dimeric arrangement with the ß-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members.

Structural Basis for Recognition of the FLAG-tag by Anti-FLAG M2.

The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 310 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody.

Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing.

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry-based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies.

Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL).

Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane-permeable disuccinimidyl suberate crosslinker, proteins on the EV outer-surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane-enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high-resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

Glycan shield of the ebolavirus envelope glycoprotein GP.

The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans.

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins.

Design and structural characterisation of olfactomedin-1 variants as tools for functional studies.

BACKGROUND: Olfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 "V" each consist of two C-terminal ß-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements. RESULTS: Here we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 ß-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform. CONCLUSIONS: These recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.

Nogo Receptor crystal structures with a native disulfide pattern suggest a novel mode of self-interaction.

The Nogo Receptor (NgR) is a glycophosphatidylinositol-anchored cell-surface protein and is a receptor for three myelin-associated inhibitors of regeneration: myelin-associated glycoprotein, Nogo66 and oligodendrocyte myelin glycoprotein. In combination with different co-receptors, NgR mediates signalling that reduces neuronal plasticity. The available structures of the NgR ligand-binding leucine-rich repeat (LRR) domain have an artificial disulfide pattern owing to truncated C-terminal construct boundaries. NgR has previously been shown to self-associate via its LRR domain, but the structural basis of this interaction remains elusive. Here, crystal structures of the NgR LRR with a longer C-terminal segment and a native disulfide pattern are presented. An additional C-terminal loop proximal to the C-terminal LRR cap is stabilized by two newly formed disulfide bonds, but is otherwise mostly unstructured in the absence of any stabilizing interactions. NgR crystallized in six unique crystal forms, three of which share a crystal-packing interface. NgR crystal-packing interfaces from all eight unique crystal forms are compared in order to explore how NgR could self-interact on the neuronal plasma membrane.

Structural basis of myelin-associated glycoprotein adhesion and signalling.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin-axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified-N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site-that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin-axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.