Biology subjects

Harris, N. J.

Publications and source records attributed to Harris, N. J..

8 recordsLinked to original sources

bioRxiv2024

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

biochemistry↗

bioRxiv2024

Molecular basis for plasma membrane recruitment of PI4KA by EFR3

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIII/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease. One sentence summaryThe cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA complex reveals the molecular mechanism underlying PI4KA membrane localisation, and provides novel insight into PI4KA regulation and its involvement in human disease.

biochemistry↗

bioRxiv2024

Structure of Calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A

Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the PI4P and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNA{beta}1 isoform of the phosphatase Calcineurin. We previously identified that CNA{beta}1 directly binds to FAM126A. Here, we report a cryo-EM structure of a truncated PI4KA complex bound to Calcineurin, revealing a direct Calcineurin interaction with PI4KA. Additional HDX-MS and computational analysis show that Calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KAs horn domain. We also characterised conserved LTLT and PSISIT Calcineurin binding sequences in the C-terminus of FAM126A. These sites are in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of Calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how Calcineurin can regulate PI4KA activity.

biochemistry↗

bioRxiv2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3K{gamma} is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKC{beta} activated by the IgE receptor, and G{beta}{gamma} subunits released from activated GPCRs. PI3K{gamma} can form two distinct complexes, with the p110{gamma} catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110{gamma} in regulating lipid kinase activity of distinct PI3K{gamma} complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110{gamma} membrane recruitment or Ras/G{beta}{gamma} binding, but instead decreased ATP turnover. We also identified that p110{gamma} can be activated by dual PKC{beta} helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKC{beta} phosphorylation is selective for p110{gamma}-p84 compared to p110{gamma}-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKC{beta} mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110{gamma} that is distinct between p110{gamma}-p84 and p110{gamma}-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

biochemistry↗

bioRxiv2021

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3K{gamma}, is a heterodimeric complex composed of a catalytic p110{gamma} subunit bound to a p101 or p84 regulatory subunit. PI3K{gamma} is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K{gamma} single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3K{gamma} regulation and identifies sites that may be exploited for therapeutic development. Highlights- HDX-MS rapidly identifies epitopes of camelid single-chain nanobodies raised against Class IB PI3K complexes, p110{gamma}/p101 and p110{gamma}/p84 - A nanobody targeting p101 improves local resolution in EM studies with p110{gamma}/p101 facilitating structural characterization of the complex - Nanobodies that bind at the interfaces with the lipidated activators Ras and G{beta}{gamma} can prevent activation of p110{gamma}/p101 and p110{gamma}/p84

biochemistry↗

bioRxiv2021

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

biochemistry↗

bioRxiv2020

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to selective inhibitors

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the p110{gamma} subunit playing a key role in immune signalling. PI3K{gamma} is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the c-terminus can inactivate or activate enzyme activity. Screening of small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3K{gamma} kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

biochemistry↗

bioRxiv2020

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

biochemistry↗