Time-Resolved Mn2+–NO and NO–NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membrane. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO–NO, Mn2+–NO, and Mn2+–Mn2+ pulsed electron-electron double resonance spectroscopy of TmrAB in a time resolved manner. This type IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+–ATP and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process

Legionella pneumophila macrophage infectivity potentiator protein appendage domains modulate protein dynamics and inhibitor binding

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires' disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein (LpMIP) have additional appendage domains of mostly unknown function. In full-length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 Å, respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Conformational flexibility of the protein insertase BamA in the native asymmetric bilayer elucidated by ESR spectroscopy

The β-barrel assembly machinery (BAM) consisting of the central β-barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins. BamA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron-electron double resonance spectroscopy to characterize BamA in the native outer membrane. In the detergent micelles, the data is consistent with mainly an inward-open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity. The lateral gate and the extracellular loop 3 exist in an equilibrium between different conformations. The outer membrane provides a favorable environment for occupying multiple conformational states independent of the lipoproteins. Our results reveal a highly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of a membrane protein in situ

Gd³⁺–Trityl–Nitroxide Triple Labeling and Distance Measurements in the Heterooligomeric Cobalamin Transport Complex in the Native Lipid Bilayers

Increased efforts are being made for observing proteins in their native environments. Pulsed electron–electron double resonance spectroscopy (PELDOR, also known as DEER) is a powerful tool for this purpose. Conventionally, PELDOR employs an identical spin pair, which limits the output to a single distance for monomeric samples. Here, we show that the Gd3+–trityl–nitroxide (NO) three-spin system is a versatile tool to study heterooligomeric membrane protein complexes, even within their native membrane. This allowed for an independent determination of four different distances (Gd3+–trityl, Gd3+–NO, trityl–NO, and Gd3+–Gd3+) within the same sample. We demonstrate the feasibility of this approach by observing sequential ligand binding and the dynamics of complex formation in the cobalamin transport system involving four components (cobalamin, BtuB, TonB, and BtuF). Our results reveal that TonB binding alone is sufficient to release cobalamin from BtuB in the native asymmetric bilayers. This approach provides a potential tool for the structural and quantitative analysis of dynamic protein–protein interactions in oligomeric complexes, even within their native surroundings

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron–electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment

Expanding Magnetic Resonance Techniques into Live Whole Cells for Membrane Protein Studies

Membrane proteins may be influenced by environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structure in a native environment are highly desirable. Whole cell DEER studies represent a novel general strategy for precise distance measurements on outer membrane proteins in whole E. coli cells and isolated outer membranes. In this work, the cobalamin transporter BtuB was overexpressed and spin labelled in whole cells and outer membranes, and interspin distances were measured to a spin labelled cobalamin using pulse EPR. A comparative analysis of the data reveals a similar interspin distance distribution between whole cells, outer membranes and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions for large outer membrane protein complexes in whole cells and native membranes, and provides a method to validate high-resolution structures of membrane proteins in their native environment. In this study, the concentration of BtuB used corresponds to about 105 copies per cell, which is comparable to the expression level of some endogenous outer membrane proteins. A comparison of data between three different membrane environments indicates that the lipid composition does not significantly influence the loop conformation. These native preparations provide a versatile tool to study membrane proteins and unlike the alternate approaches, does not require the tedious processes of solubilization, purification, and membrane reconstitution of the target protein