Mitochondrial ADP/ATP Carrier in Dodecylphosphocholine Binds Cardiolipins with Non-native Affinity

Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of docecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins (CLs) is preserved in DPC, thereby suggesting that DPC is a suitable environment to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of CLs to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investiga- tion in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment

Evaluating the motional timescales contributing to averaged anisotropic interactions in MAS solid-state NMR

Dynamic processes in molecules can occur on a wide range of timescales, and it is important to understand which timescales of motion contribute to different parameters used in dynamics measurements. For spin relaxation, this can easily be understood from the sampling frequencies of the spectral-density function by different relaxation-rate constants. In addition to data from relaxation measurements, determining dynamically averaged anisotropic interactions in magic-angle spinning (MAS) solid-state NMR allows for better quantification of the amplitude of molecular motion. For partially averaged anisotropic interactions, the relevant timescales of motion are not so clearly defined. Whether the averaging depends on the experimental methods (e.g., pulse sequences) or conditions (e.g., MAS frequency, magnitude of anisotropic interaction, radio-frequency field amplitudes) is not fully understood. To investigate these questions, we performed numerical simulations of dynamic systems based on the stochastic Liouville equation using several experiments for recoupling the dipolar coupling, chemical-shift anisotropy or quadrupolar coupling. As described in the literature, the transition between slow motion, where parameters characterizing the anisotropic interaction are not averaged, and fast motion, where the tensors are averaged leading to a scaled anisotropic quantity, occurs over a window of motional rate constants that depends mainly on the strength of the interaction. This transition region can span 2 orders of magnitude in exchange-rate constants (typically in the microsecond range) but depends only marginally on the employed recoupling scheme or sample spinning frequency. The transition region often coincides with a fast relaxation of coherences, making precise quantitative measurements difficult. Residual couplings in off-magic-angle experiments, however, average over longer timescales of motion. While in principle one may gain information on the timescales of motion from the transition area, extracting such information is hampered by low signal-to-noise ratio in experimental spectra due to fast relaxation that occurs in the same region.</p

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT (“Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301“), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, “Investing in your future”). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017)

Deuteration of proteins boosted by cell lysates: high-resolution amide and H<i>α</i> magic-angle-spinning (MAS) NMR without the reprotonation bottleneck

Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.</p

Characterization of Imaging Luminance Measurement Devices (ILMDs)

CIE 244:2021This document describes the elements, function and characterization of imaging luminance measuring devices (ILMDs). Furthermore, the calibration of ILMDs is described and some guidelines for their use are provided. Using ILMDs the projection of the luminance distribution of a scene can be recorded and made available for further evaluation. In addition to a simple documentation of measurements, the geometrical assignment of the image points into the object coordinate system often allows more complex calculations by combining luminance, directional and, if necessary, solid angle information (e.g. for glare evaluation). In addition to the flexible evaluation option, it is possible to acquire a large number of measured values quickly and, if necessary, even synchronously. Furthermore, the type of evaluation can also be coupled to the image content, i.e. the image areas to be evaluated can be determined in the image either by their position within the image or by their luminance value

In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-Synuclein within E. coli cells

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson’s disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution

Molecular basis of FIR-mediated c-myc transcriptional control

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.MRC Grant-in-aid U11757455