Malaria kills over 400,000 people each year and nearly half the world's population live in at-risk areas. Progress against malaria has recently stalled, highlighting the need for developing novel ...therapeutics. The parasite haemoglobin degradation pathway, active in the blood stage of the disease where malaria symptoms and lethality manifest, is a well-established drug target. A key enzyme in this pathway is the papain-type protease falcipain-2.
The crystallographic structure of falcipain-2 at 3.45 Å resolution was resolved in complex with an (E)-chalcone small-molecule inhibitor. The falcipain-2-(E)-chalcone complex was analysed with reference to previous falcipain complexes and their similarity to human cathepsin proteases.
The (E)-chalcone inhibitor binds falcipain-2 to the rear of the substrate-binding cleft. This is the first structure of a falcipain protease where the rear of the substrate cleft is bound by a small molecule. In this manner, the (E)-chalcone inhibitor mimics interactions observed in protein-based falcipain inhibitors, which can achieve high interaction specificity.
This work informs the search for novel anti-malaria therapeutics that target falcipain-2 by showing the binding site and interactions of the medically privileged (E)-chalcone molecule. Furthermore, this study highlights the possibility of chemically combining the (E)-chalcone molecule with an existing active-site inhibitor of falcipain, which may yield a potent and selective compound for blocking haemoglobin degradation by the malaria parasite.
The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA ...assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
Correct folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the β-barrel assembly machinery (BAM). How unfolded ...substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA β1-β16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.
Thermocouples are widely used in industry, but a commonly encountered difficulty is the thermoelectric drift which causes an unknown change to the thermovoltage over time. A comprehensive survey of ...the published information on drift rates for Types B, C, D, K, N, R, S, Au/Pt, and Pt/Pd thermocouples is presented. The data that are available for thermocouple drift rates are reviewed for each major thermocouple type, and any large gaps in the reported data are stated. The effects of different parameters on the drift rate are summarized, with a particular focus on what happens at different temperatures. The effects of temperature, thermoelement diameter, sheath and/or insulation type, atmosphere, and thermocycling on the drift rate are considered (where data are available). This will allow users to assess the likely magnitude of the drift rate of different thermocouples for a given application.
Tau and Aβ assemblies of Alzheimer’s disease (AD) can be visualized in living subjects using positron emission tomography (PET). Tau assemblies comprise paired helical and straight filaments (PHFs ...and SFs). APN-1607 (PM-PBB3) is a recently described PET ligand for AD and other tau proteinopathies. Since it is not known where in the tau folds PET ligands bind, we used electron cryo-microscopy (cryo-EM) to determine the binding sites of APN-1607 in the Alzheimer fold. We identified two major sites in the β-helix of PHFs and SFs and a third major site in the C-shaped cavity of SFs. In addition, we report that tau filaments from posterior cortical atrophy (PCA) and primary age-related tauopathy (PART) are identical to those from AD. In support, fluorescence labelling showed binding of APN-1607 to intraneuronal inclusions in AD, PART and PCA. Knowledge of the binding modes of APN-1607 to tau filaments may lead to the development of new ligands with increased specificity and binding activity. We show that cryo-EM can be used to identify the binding sites of small molecules in amyloid filaments.
The β‐barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram‐negative bacteria, and represents a promising antimicrobial target. Several conformational ...states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron‐Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR‐B), BAM′s conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in‐cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR‐B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
The darobactins are promising new antibiotics that inhibit the BAM complex, an essential membrane‐protein foldase in Gram‐negative bacteria. Here, we use EPR spectroscopy (PELDOR, or DEER) distance measurements to monitor the effect of darobactin B on BAM in E. coli cells, showing it stabilises BAM in a lateral gate closed conformational state. Our results provide the first direct evidence for conformational selection by a darobactin in the cellular context.
Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic ...biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo 3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo 3 and can also be performed with complex membrane protein mixtures.
The β‐barrel assembly machine (BAM) is essential for folding outer membrane proteins (OMPs) into the outer membrane (OM) of Gram‐negative bacteria. Structures of BAM have been solved using X‐ray ...crystallography and cryoEM, but the conformation of BAM in the highly crowded native OM remained unsolved. Using EPR spectroscopy of BAM in intact E. coli cells, the structure of BAM in situ has now been revealed in the Research Article (e202218783) by Sheena E. Radford, Christos Pliotas et al., both alone and in the presence of the antibacterial, darobactin B. Image: Jeroen Claus (Phospho Biomedical Animation).
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