Biology subjects

Byron, O.

Publications and source records attributed to Byron, O..

5 recordsLinked to original sources

bioRxiv2024

AdhE spirosome length in enterohaemorrhagic Escherichia coli is correlated with enzymatic directionality and is perturbed by salicylidene acylhydrazide binding

Antibiotics are contraindicated for the treatment of infection enterohemorrhagic Escherichia coli (EHEC), a human pathogen that causes diarrhea or hemorrhagic colitis in humans which can progress to hemolytic uremic syndrome (HUS). As an alternative to the use of antibiotics, previous studies developed the salicylidene acylhydrazides (SA), a family of anti-virulence compounds capable of blocking expression of the type three secretion system (T3SS), thereby reducing bacterial infections. Here we validate AdhE as the anti-virulence protein target of the SA compound ME0054. AdhE is a bidirectional enzyme able to catalyse the conversion of acetyl-CoA to ethanol and vice versa. AdhE oligomerises forming helicoidal filaments heterogeneous in length called spirosomes. In this work, we show that it is possible to partially fractionate AdhE spirosomes using size exclusion chromatography (SEC) and to characterise the spirosome oligomers present in each fraction with biophysical techniques such as small angle X-ray scattering (SAXS) and sedimentation velocity analytical ultracentrifugation (SV-AUC). Also, we observe that short spirosomes are more efficient in the reverse reaction whereas the spirosome length has no impact on the forward reaction. Therefore, for first time, we reveal that AdhE spirosome formation is necessary to regulate the direction of its enzymatic reactions. In addition, we show that ME0054 disrupts AdhE spirosomes, thereby enhancing the conversion of ethanol to acetyl-CoA. Importantly, SV-AUC data show that ME0054 binds to the AdhE filaments. Finally, time-resolved (TR) SAXS allowed us to follow the kinetics of spirosome disruption produced by ME0054, confirming its effectiveness at biologically relevant temperatures and timescales. SIGNIFICANCE STATEMENTThere is an urgent need to develop alternative strategies to combat bacterial infections. Salicylidene acylhydrazides (SA) are able to reduce expression of the bacterial type three secretion system (T3SS), used by many pathogens to manipulate host eukaryotic cells, including our pathogen of interest: enterohaemorrhagic E. coli (EHEC). The mechanism underpinning these compounds is a mystery. Here we show how the SA compound ME0054, by disrupting AdhE spirosomes, enhances metabolic conversion of ethanol to acetyl-CoA. This finding is consistent with the phenotype observed in an EHEC AdhE mutant: alterations in acetate levels and changes in T3SS expression. Our work establishes a crucial mechanistic link between the binding of the SA compound to a key target protein and changes in bacterial metabolism.

biophysics↗

bioRxiv2021

Non-canonical Staphylococcus aureus pathogenicity island repression

Mobile genetic elements (MGEs) control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of the Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation, never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature. SignificanceWhile most repressors controlling the transfer of mobile genetic elements are dimers, we demonstrate here that the Staphylococcal pathogenicity island 1 (SaPI1) is repressed by two tetramers, which have a novel structural fold in their body that has never been seen before in other proteins. Moreover, by solving the structure of the SaPI1 repressor in complex with its inducing protein Sri, we have demonstrated that Sri forces the SaPI1 repressor to adopt a conformation that is incompatible with DNA binding, explaining how SaPI1 is induced. Finally, our results demonstrate that this repression system is not exclusive of the SaPIs but widespread in nature. Our studies provide important insights understanding how SaPIs spread in nature.

molecular biology↗

bioRxiv2019

Structural Basis for CAL1-Mediated Centromere Maintenance

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting is achieved by HJURP and the Mis18 complex, respectively. Here, using crystal structures, we show how Drosophila CAL1, an evolutionarily distinct CENP-A chaperone, targets CENP-A to the centromere receptor CENP-C without the requirement of the Mis18 complex: while the N-terminal CAL1 fragment (CAL11-160) wraps around CENP-A/H4 through multiple physical contacts, the C-terminal CAL1 fragment (CAL1893-914) directly binds CENP-C cupin dimer. Our work shows CAL1, though divergent at the primary structure, employs evolutionarily conserved and adaptive structural principles to recognise CENP-A/H4 and CENP-C providing insights into the minimalistic principles underlying centromere maintenance.

biochemistry↗

bioRxiv2018

Calicivirus VP2 forms a portal to mediate endosome escape

To initiate the infectious process, many viruses enter their host cells by triggering endocytosis following receptor engagement. The mechanism by which non-enveloped viruses, such as the caliciviruses, escape the endosome is however poorly understood. The Caliciviridae include many important human and animal pathogens, most notably norovirus, the cause of winter vomiting disease. Here we show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal assembly at a unique three-fold symmetry axis following receptor engagement. This feature surrounds an open pore in the capsid shell. We hypothesise that the VP2 portal complex is the means by which the virus escapes the endosome, pene-trating the endosomal membrane to release the viral genome into the cytoplasm. Cryogenic electron microscopy (cryoEM) and asymmetric reconstruction were used to investigate structural changes in the capsid of feline calicivirus (FCV) that occur when the virus binds to its cellular receptor junctional adhesion molecule-A (fJAM-A). Near atomic-resolution structures were calculated for the native virion alone and decorated with soluble receptor fragments. We present atomic models of the major capsid protein VP1 in the presence and absence of fJAM-A, revealing the contact interface and conformational changes brought about by the interaction. Furthermore, we have calculated an atomic model of the portal protein VP2 and revealed the structural changes in VP1 that lead to pore formation. While VP2 was known to be critical for the production of infectious virus, its function has been hitherto undetermined. Our finding that VP2 assembles a portal that is likely responsible for endosome escape represents a major step forward in our understanding of both the Caliciviridae and icosahedral RNA containing viruses in general.

microbiology↗

bioRxiv2018

Merging in-solution X-ray and neutron scattering data allows fine structural analysis of membrane-protein detergent complexes

AbstractIn-solution small angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labelled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. Firstly, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS which has been adapted to predict SANS data. Secondly, the use of MONSA which allows to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/324103_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (67K):\norg.highwire.dtl.DTLVardef@f7e17corg.highwire.dtl.DTLVardef@1f4987dorg.highwire.dtl.DTLVardef@1475c79org.highwire.dtl.DTLVardef@839b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗