Biology subjects

Ahdash, Z.

Publications and source records attributed to Ahdash, Z..

6 recordsLinked to original sources

bioRxiv2025

Molecular basis for multidrug efflux by an anaerobic RND transporter

Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. We reveal that drug binding domain and channel conformational plasticity likely governs promiscuous substrate specificity, analogous to its closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Physiologically, this provides means of xenobiotic and metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract.

biochemistry↗

bioRxiv2020

The allosteric modulation of Complement C5 by knob domain peptides

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 [A] from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

immunology↗

bioRxiv2020

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-{beta}-naphthylamide (PA{beta}N) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

biochemistry↗

bioRxiv2019

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

biophysics↗

bioRxiv2018

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.\n\nOne sentence summaryStructure and dynamics of the CSN-CRL2 complexes assessed by cryo-electron microscopy and structural mass spectrometry.

biochemistry↗

bioRxiv2018

Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry

SummaryHydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS.\n\nAvailabilityDeuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license.\n\nImplementationwritten in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library.\n\nContactargyris.politis@kcl.ac.uk

bioinformatics↗