Biology subjects

Calabrese, A. N.

Publications and source records attributed to Calabrese, A. N..

18 recordsLinked to original sources

bioRxiv2026

Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

Deubiquitylases modulate cellular processes by cleaving monoubiquitin or polyubiquitin chains. The ARISC-RAP80 complex partners with BRCA1-BARD1 to form the BRCA1-A super-complex, which recognizes K63-linked ubiquitin chains at DNA damage sites. ARISC-RAP80 contains multiple ubiquitin-binding sites, yet how these influence recognition and cleavage of K63-polyubiquitylated substrates remains unknown. We discover that a composite three-subunit interface allows ARISC-RAP80 to position K63-linked polyubiquitin chains in its catalytic site. Substrate recognition is further supported by RAP80 and non-catalytic ubiquitin-binding sites that impose a compact conformation to K63-polyubiquitylated substrates. This mechanism exploits the inherent flexibility of long ubiquitin chains and differs considerably from other deubiquitylases. Structure-guided mutagenesis validate ubiquitin chain interactions, and cell-based assays demonstrate a functional role of the observed interfaces in chromatin recruitment. Our findings define mechanisms of polyubiquitin chain decoding and cleavage by ARISC-RAP80, linking ubiquitin reading and erasing functions to BRCA1-A mediated DNA damage responses.

biochemistry↗

bioRxiv2026

Cross-Platform Assessment of Sub-50 nm Nanopipette Emitters for Native Electrospray Ionization Mass Spectrometry

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein range (14.5-800 kDa). We analysed the intrinsically disordered protein alpha synuclein ( S; 14.5 kDa) and holo-myoglobin (17 kDa) over a range of concentrations (10 M-1 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observed reduced Na adduction and preservation of the Zn 2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (~44 kDa), malate dehydrogenase (~70 kDa), transferrin (80 kDa), glutamate dehydrogenase (~350 kDa), {beta}-galactosidase (~465 kDa), and GroEL (~800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 m internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing -synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

biochemistry↗

bioRxiv2025

Unveiling the unique interaction mechanism of herpes simplex virus 2 glycoprotein C with C3b

AbstractThe complement cascade is part of the first line of defence against viral infections, and many viruses have evolved to block it. For example, glycoprotein C (gC) from Herpes Simplex Virus 1 and 2 (gC1 and gC2) facilitates infection by modulating the complement cascade through an interaction with C3b. gC is also involved in attachment and other viral processes. However, our understanding of the molecular mechanisms of gC have been limited due to the absence of a structure. AlphaFold predicts that gC contains a disordered N-terminus and three immunoglobulin-like domains. Here, we generated various gC2 constructs and demonstrated that gC2 domains 1 and 2 are necessary and sufficient to interact with C3b and block the alternative pathway. A gC2 construct lacking the N-terminus in complex with C3b was characterised by cryo-EM at 3.6 [A], providing the first structure for gC2, and revealing that the interaction is predominantly driven by gC2 domain 2 and the MG8 domain of C3b. This structure was confirmed by cross-linking mass spectrometry and by using C3b-blocking antibodies that recognised gC2 linear epitopes at the interface with C3b. Overall, the gC-C3b interaction is different from other C3b-interacting partners, providing a novel mechanism to regulate the complement cascade.

molecular biology↗

bioRxiv2025

Buffer-dependent conformational dynamics of α-synuclein revealed by nanopipette electrospray ionisation ion mobility mass spectrometry

Electrolyte conditions in vivo and in vitro are known to influence protein structure and function. Intrinsically disordered proteins (IDPs) are particularly sensitive to their solution conditions such as ionic strength and molecular crowding, and their dynamic structural ensembles rapidly respond to the cellular environment. While structural mass spectrometry (MS) techniques are uniquely able to capture aspects of this structural diversity, technical limitations have largely precluded the use of native MS approaches to interrogate the conformational rearrangements of IDPs in response to high concentrations of non-volatile salts. Here, we overcome this challenge by employing sub 100-nm nanopipette electrospray emitters for more gentle and salt-tolerant analysis to study the conformational distribution of -Synuclein (S) using native MS and ion mobility-MS in varied solution conditions, including in phosphate buffered saline. We show using native MS that it is possible to capture salt and buffer induced changes in the S conformational ensemble when using traditional biochemical buffers, which reflect structural changes from in silico predictions and in-solution measurements. This work demonstrates the power of nanopipette emitters for the study of IDPs, and establishes native MS as a method that can be routinely used to determine how solution conditions tune the conformational landscape of IDPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/671163v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@35ff69org.highwire.dtl.DTLVardef@113821aorg.highwire.dtl.DTLVardef@1c27b90org.highwire.dtl.DTLVardef@13e4db1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2025

Design and Characterisation of Photoactivatable and Lysine Reactive o-Nitrobenzyl Alcohol-Based Crosslinkers

Photoreactive groups are invaluable tools in structural proteomics, offering reagent-free activation and temporal control of protein labelling. However, traditional UV-activatable functional groups often produce unstable intermediates and diverse products, making these chemistries difficult to deploy at scale. In this study, we performed a systematic analysis of ortho-nitrobenzyl alcohol (oNBA) reactivity for integration into novel reagents for chemical crosslinking-mass spectrometry. oNBA photochemistry represents a promising alternative to traditional photoactivatable crosslinkers due to its unique specificity towards lysine residues. Here, we synthesised two molecules comprising oNBA functional groups with different substituents and assessed their labelling efficiency against a model protein. To ensure high labelling yields while maintaining a short irradiation time, we constructed a high power 365 nm irradiation device which improves the efficiency of oNBA photolysis. Our studies identified an amide-substituted probe that labels proteins with high efficiency. We next incorporated this optimised oNBA moiety into a homo-bifunctional crosslinker and a hetero-bifunctional crosslinker in combination with an NHS ester, which both resulted in high yields of crosslinked products. Our findings highlight that optimised oNBA-based reactive groups are viable UV-activated warheads that can deliver high labelling yields and efficient protein crosslinking, unlocking a wealth of potential structural proteomics applications.

biophysics↗

bioRxiv2024

Mechanism of Interaction Between the Transactivation Domain of N-MYC and the DNA-Binding Surface of TFIIIC5

N-myc is a member of the myc family of transcription factors, which are powerful drivers of cellular growth and consequently, important oncoproteins. N-myc interacts with many factors and complexes to affect transcription. One such complex is the RNA Polymerase III assembly factor, TFIIIC, a six-member complex that is essential for the transcription of small, structured RNA. TFIIIC and N-myc mutually restrict each others chromatin association, and their complex contributes to quality control in mRNA transcription. We previously demonstrated that the largely intrinsically disordered transactivation domain of N-myc interacts directly with a sub-complex of TFIIIC, {tau}A. Structural studies by others show that DNA binding of {tau}A is largely mediated by TFIIIC3, which suggests that TFIIIC5 is at most a secondary binding site for DNA. Here we identified the DNA binding domain of TFIIIC5 as a key binding site for N-myc. We used an integrated approach combining NMR, HDX mass spectrometry, pull-downs and biophysical assays to elucidate the molecular basis of the interaction. Two sequences in the transactivation domain of N-myc bind to the DNA binding interface of TFIIIC5. AlphaFold modelling predicts a high-confidence binding mode for the higher affinity N-myc motif that overlaps with the predicted intramolecular binding site of the C-terminal acidic plug of TFIIIC5, removal of which enhances the binding of N-myc. The same two motifs in N-myc also interact with Aurora-A kinase, which competes with N-myc for TFIIIC binding during S-phase. This model elucidates how the N-myc:TFIIIC5 interaction competes with other interactions, providing a basis for their mutual censoring function and regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/619198v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e4a76aorg.highwire.dtl.DTLVardef@ab349corg.highwire.dtl.DTLVardef@af85dorg.highwire.dtl.DTLVardef@37dd2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

biochemistry↗

bioRxiv2024

Structural characterization and inhibition of the interaction between ch-TOG and TACC3

The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating alone, together or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the -helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3-ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells following the formation of a bipolar spindle and delayed mitotic progression; uncovering a novel role of TACC3-ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

cell biology↗

bioRxiv2024

Affinity purification mass spectrometry characterization of the interactome of receptor tyrosine kinase proline rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40% of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

cell biology↗

bioRxiv2023

The structural architecture of an α-synuclein toxic oligomer

Oligomeric species populated during -synuclein aggregation are considered key drivers of neurodegeneration in Parkinsons disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. PSM3 is a nanomolar peptide binder of -synuclein oligomers that inhibits aggregation by blocking oligomer to fibril conversion. Here, we investigate the binding of PSM3 to -synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSM3 selectively targets an -synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the monomeric and oligomeric states. This -synuclein region plays a pivotal role in oligomer to fibril conversion, as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early-onset Parkinsons disease, causes a conformational fluctuation in this region, leading to delayed oligomer to fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in -synuclein oligomers, advance our comprehension of oligomer to amyloid fibril conversion and reveal a new facet of -synuclein pathogenic mutations.

biophysics↗

bioRxiv2022

An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2

The ability of broadly neutralising antibodies to target conserved epitopes gives them huge potential as antibody-based therapeutics, particularly in the face of constant viral antigen evolution. Certain bovine antibodies are highly adept at binding conserved, glycosylated epitopes, courtesy of their ultralong complementarity determining region (CDR)H3. Here, we used a SARS-naive, bovine ultralong CDRH3 library and mammalian cell display, to isolate a bovine paratope that engages the SARS-CoV and SARS-CoV-2 receptor-binding domain (RBD). This neutralises viruses pseudo-typed with SARS-CoV Spike protein but not by competition with RBD binding to ACE2. Instead, using differential hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis, we demonstrate that this ultralong CDRH3 recognises a rarely identified, conserved, cryptic epitope that overlaps the target of pan-sarbecovirus antibodies (7D6/6D6). The epitope is glycan-shielded and becomes accessible only transiently via inter-domain movements. This represents the first bovine anti-sarbecovirus paratope and highlights the power of this approach in identifying novel tools to combat emerging pathogens.

molecular biology↗

bioRxiv2021

Mechanical and molecular activation lead to structurally analogous MscL states

The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.

biophysics↗

bioRxiv2020

Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between eleven distinct RNA molecules, all of which must be selectively co-packaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 auto-regulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, whilst accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch poised towards RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation whilst promoting inter-molecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

biophysics↗

bioRxiv2020

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yields inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly-populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualisation of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.

biochemistry↗

bioRxiv2019

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperones domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

biochemistry↗

bioRxiv2019

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

The {beta}-barrel assembly machinery (BAM) inserts outer membrane {beta}-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Although BAM has been the focus of intense research due to its essential activity in generating and maintaining the outer membrane, how it functions remains poorly understood. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. The globular domain of RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. Our structural and biochemical data indicate a push-and-pull mechanism for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. Overall, the structural insights gleaned here elucidate a fundamental biological process and suggest a new avenue for antibiotic development.

microbiology↗

bioRxiv2018

Stability of local secondary structure determines selectivity of viral RNA chaperones

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus NSP2 and avian reovirus {sigma}NS. Here, we compared the abilities of NSP2 and {sigma}NS to mediate sequence-specific interactions between rotavirus genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while {sigma}NS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric {sigma}NS oligomerises into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

biophysics↗