Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

Spin-Labelled Mechanically Interlocked Molecules as Models for the Interpretation of Biradical EPR Spectra

<div> <p>Biradical spin probes can provide detailed information about the distances between molecules/regions of molecules because the through-space coupling of radical centres, characterised by <i>J</i>, is strongly distance dependent. However, if the system can adopt multiple configurations, as is common in supramolecular complexes, the shape of the EPR spectrum is influenced not only by <i>J</i> but also the rate of exchange between different states. In practice, it is often hard to separate these variables and as a result, the effect of the latter is sometimes overlooked. To demonstrate this challenge unequivocally we synthesised rotaxane biradicals containing nitronyl nitroxide units at the termini of their axles. The rotaxanes exchange between the available biradical conformations more slowly than the corresponding non-interlocked axles but, despite this, in some cases, the EPR spectra of the axle and rotaxane remain remarkably similar.<i> </i>Detailed analysis allowed us to demonstrate that the similar EPR spectral shapes result from different combinations of <i>J</i> and rates of conformational interconversion, a phenomenon suggested theoretically more than 50 years ago. This work reinforces the idea that thorough analysis must be performed when interpreting the spectra of biradicals employed as spin probes in solution.</p></div>

Spin-Labelled Mechanically Interlocked Molecules as Models for the Interpretation of Biradical EPR Spectra

<div> <p>Biradical spin probes can provide detailed information about the distances between molecules/regions of molecules because the through-space coupling of radical centres, characterised by <i>J</i>, is strongly distance dependent. However, if the system can adopt multiple configurations, as is common in supramolecular complexes, the shape of the EPR spectrum is influenced not only by <i>J</i> but also the rate of exchange between different states. In practice, it is often hard to separate these variables and as a result, the effect of the latter is sometimes overlooked. To demonstrate this challenge unequivocally we synthesised rotaxane biradicals containing nitronyl nitroxide units at the termini of their axles. The rotaxanes exchange between the available biradical conformations more slowly than the corresponding non-interlocked axles but, despite this, in some cases, the EPR spectra of the axle and rotaxane remain remarkably similar.<i> </i>Detailed analysis allowed us to demonstrate that the similar EPR spectral shapes result from different combinations of <i>J</i> and rates of conformational interconversion, a phenomenon suggested theoretically more than 50 years ago. This work reinforces the idea that thorough analysis must be performed when interpreting the spectra of biradicals employed as spin probes in solution.</p></div>

Stabilization of glyphosate zwitterions and conformational/tautomerism mechanism in aqueous solution: insights from ab initio and density functional theory-continuum model calculations

Theoretical investigation of the conformational interconversion equilibria and the ZW ⇋ NE tautomerization process of glyphosate in an aqueous solution using IEFPCM and SMD continuum solvation models.

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Determination of the cationic conformational structure of tetrahydrothiophene by one-photon MATI spectroscopy and Franck–Condon fitting

This study affords more accurate information regarding the pseudorotational PES describing the conformational interconversion of THT.