Biology subjects

Calvaresi, V.

Publications and source records attributed to Calvaresi, V..

6 recordsLinked to original sources

bioRxiv2026

Allosteric remodelling of the Ebola virus glycoprotein underlies differences in entry mechanisms across species

During Ebolavirus transit through the endo-lysosomal pathway, the glycoprotein GP undergoes a series of conformational rearrangements upon cathepsin cleavage and NPC1 receptor binding, culminating in host-virus membrane fusion. While these rearrangements underpin viral entry, their molecular mechanisms remain poorly understood. Here, we combine hydrogen/deuterium-exchange mass spectrometry and mass photometry to resolve structural dynamic and energetic transitions of GP of two major species, Zaire (EBOV) and Sudan (SUDV). We describe the allosteric axes that govern reorganization of the cleavage sites, opening of the receptor binding cavity and priming for fusion upon NPC1 engagement. We show that GP cathepsin cleavage and receptor binding exhibit different mechanisms and kinetics across species, and are allosterically coupled in a species-specific manner. We reveal that fusion priming is optimized via distinct routes in EBOV and SUDV GP. These data suggest that GP structural dynamics and allosteric remodelling underlie differences in entry mechanisms across Ebolavirus species.

biochemistry↗

bioRxiv2026

LemonCatcher Acidic Pull-Down Enables Selective In-Cell Hydrogen-Deuterium Exchange Mass Spectrometry

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

biochemistry↗

bioRxiv2025

An Evolutionary Distinct Nipah G Glycosylation Site Provides Stability for Receptor Engagement

Nipah virus is a deadly paramyxovirus with 40-75% mortality and >750 cases since 1998. Currently there are no clinically approved vaccines or therapeutics to target infection. Nipah is an enveloped virus with two surface glycoproteins, the trimeric fusion (F) and tetrameric attachment glycoprotein (G). G is responsible for cellular attachment via binding to ephrin B2/B3. Glycosylation of Nipah G and its effects on receptor engagement has not previously been studied but is important as glycosylation impacts immunogenicity, receptor binding and structural conformations for other enveloped virus glycoproteins. Our phylogenetic and mass spectrometry analysis of site-specific N-glycans of the Nipah G Malaysia strain revealed how N-glycosylation has evolved since the appearance of the virus in 1998. We discovered that the N481 N-glycosite is not conserved and although the glycan does not directly contribute to receptor binding, the threonine/serine in the glycosylation sequon is critical for maintaining long-range stability of individual G subunits that facilitates ephrin B2 binding affinity. Together, these data reveal plasticity of N-glycosylation sites across Nipah species and the presence of hydrogen bonding networks that contribute to G stability and host engagement, which is valuable information for understanding virus attachment/entry mechanisms as well as the rationale design of structure-based vaccines. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/646399v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@15960a8org.highwire.dtl.DTLVardef@d8cacdorg.highwire.dtl.DTLVardef@ea6eb4org.highwire.dtl.DTLVardef@10a2c84_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Structural and Functional Glycosylation of the Abdala COVID-19 Vaccine

Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics.

biochemistry↗

bioRxiv2024

Structural dynamics and immunogenicity of the recombinant and outer membrane vesicle-embedded Meningococcal antigen NadA

The structure and conformation adopted by protein vaccine antigens significantly influence the exposure of their antigenic determinants. Structural knowledge of antigens in native state could drive the design of recombinant vaccines that resemble their cognate native forms, although such information is often difficult to obtain, particularly for membrane proteins. Here, we assessed the structural and functional features of the native Neisseria Adhesin A (NadA), a meningococcal trimeric outer membrane protein included as soluble recombinant antigen in the 4CMenB vaccine. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to generate a structural model of NadA and to compare the fold and structural dynamics of the recombinant NadA as soluble vaccine form, and the native NadA in situ, as embedded in meningococcal outer membrane vesicles (OMVs), complementing the HDX data with electron microscopy imaging. While their overall structures are similar, conformational differences between the two forms were observed. Especially, OMV- embedded NadA appears more susceptible to trimer opening than its cognate soluble antigen, suggesting that NadA in its native membrane could display a larger antigenic surface. Accordingly, we show that mice immunized with OMV-embedded NadA elicited antibodies with superior bactericidal activity and capable of better preventing bacterial adhesion compared to the soluble antigen. Collectively, these data support the hypothesis that protein vaccine antigens presented in native-like environments can elicit a more potent immune response than recombinant forms.

biophysics↗

bioRxiv2022

Integrative HDX-MS enables quantification of the conformational landscape of the sugar transporter XylE

A yet unresolved challenge in structural biology is to quantify conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilising them for in vitro studies. To address this challenge, we present here an integrative strategy that combines hydrogen-deuterium exchange mass spectrometry (HDX-MS) with ensemble modelling. We benchmark our strategy on wild type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments and identify key lipid contacts that modulate protein conformations. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles, allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modelling to capture, accurately quantify and subsequently visualise co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors. For Table of Content Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/499559v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@cf7b35org.highwire.dtl.DTLVardef@1a1da1eorg.highwire.dtl.DTLVardef@f70627org.highwire.dtl.DTLVardef@1b52b90_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗