Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses

ABSTRACTThree pathogenic human coronaviruses have emerged, with SARS-CoV-2 causing a global pandemic. While therapeutic antibodies targeting the SARS-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report three antibodies, binding epitopes conserved on the pre-fusion MERS, SARS-1 and SARS-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes in in vitro SARS-2 cell fusion and pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, consistent with binding to natural and engineered spike variants. This location at the spike trimer interface suggests 3A3 prevents the S2 conformational rearrangements required for virus-host cell fusion. This work defines a highly conserved vulnerable site on the SARS-2 S2 domain and may help guide the design of pan-protective spike immunogens.TEASERA conserved, neutralizing epitope in the S2 domain of coronavirus spike was identified as a target for pan-coronavirus therapy and vaccination.

Download Full-text

Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

10.1101/810994 ◽

2019 ◽

Author(s):

Isaac Fisher ◽

Meredith Jenkins ◽

Greg Tall ◽

John E Burke ◽

Alan V. Smrcka

Keyword(s):

Phospholipase C ◽

Inositol Phosphates ◽

G Protein Coupled Receptors ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms ◽

G Protein Coupled ◽

Inhibitory Interactions

AbstractPhospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

Download Full-text

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

Antibodies ◽

10.3390/antib8040049 ◽

2019 ◽

Vol 8 (4) ◽

pp. 49

Author(s):

Kuhne ◽

Bonnington ◽

Malik ◽

Thomann ◽

Avenal ◽

...

Keyword(s):

Conformational Changes ◽

Structural Information ◽

Binding Properties ◽

Engineering Technology ◽

Exchange Mass Spectrometry ◽

Structural Impact ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

The Impact

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure–function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

Download Full-text

Mapping the allosteric effects that define functional activity of SARS-CoV-2 specific antibodies

10.1101/2021.12.27.474251 ◽

2021 ◽

Author(s):

Nikhil K. Tulsian ◽

Palur V. Raghuvamsi ◽

Xinlei Qian ◽

Gu Yue ◽

Bhuvaneshwari D/O Shunmuganathan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Target Antigen ◽

Dynamic Features ◽

Functional Activities ◽

Human Antibodies ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Dynamics Simulations

AbstractPrevious studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies.TeaserDifferent neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein

Download Full-text

Crystal structure of an archaeal CorB magnesium transporter

Nature Communications ◽

10.1038/s41467-021-24282-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yu Seby Chen ◽

Guennadi Kozlov ◽

Brandon E. Moeller ◽

Ahmed Rohaim ◽

Rayan Fakih ◽

...

Keyword(s):

Crystal Structure ◽

Analytical Ultracentrifugation ◽

Structural Rearrangements ◽

Exchange Mass Spectrometry ◽

Magnesium Transporter ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Atp Sensing

AbstractCNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.

Download Full-text

The middle lipin domain adopts a membrane-binding dimeric protein fold

Nature Communications ◽

10.1038/s41467-021-24929-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Weijing Gu ◽

Shujuan Gao ◽

Huan Wang ◽

Kaelin D. Fleming ◽

Reece M. Hoffmann ◽

...

Keyword(s):

Protein Fold ◽

Catalytic Core ◽

Hydrophobic Residues ◽

Exchange Mass Spectrometry ◽

Co Activation ◽

Hydrogen Deuterium ◽

Lipin 1 ◽

Deuterium Exchange Mass Spectrometry ◽

Dimeric Protein

AbstractPhospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Download Full-text

The middle lipin (M-Lip) domain is a new dimeric protein fold that binds membranes

10.1101/2021.03.19.436211 ◽

2021 ◽

Author(s):

Weijing Gu ◽

Shujuan Gao ◽

Huan Wang ◽

Kaelin D Fleming ◽

Reece M Hoffmann ◽

...

Keyword(s):

Protein Fold ◽

Hydrophobic Residues ◽

Exchange Mass Spectrometry ◽

Co Activation ◽

Hydrogen Deuterium ◽

Lipin 1 ◽

Deuterium Exchange Mass Spectrometry ◽

Dimeric Protein ◽

Novel Protein

Phospholipid synthesis and fat storage as triglycerides is regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized and novel protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in full-length lipin 1 influences PAP activity, membrane binding, subcellular localization, oligomerization, and adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a new dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Download Full-text