Pauline Juyoux

Respiratory type II NADH:quinone oxidoreductases (NDH-II) are typically monotopic flavoproteins that make direct contact with the membrane to access the quinone pool. Here, we show that in Bacillus subtilis , one NDH-II, termed Ndh, assembles with the helical membrane plugin (HMP) protein YjlC and forms supramolecular fibers. Genetic and biochemical analyses demonstrate that Ndh and YjlC proteins are essential for NADH oxidation. Cryo-EM analysis reveals that YjlC forms a tubular scaffold onto which multiple Ndh subunits are regularly docked via their C-terminal domain, repurposed from its classical role in direct membrane binding. These fibers can extend up to ~1000 Å, creating a continuous hydrophobic tunnel filled with lipids and quinones, thereby mimicking the membrane environment. Comparative genomics unveils that this partnership arose exclusively within Bacillales through the recruitment of an ancestral HMP originally associated with sulfide:quinone reductases. Together, our findings uncover a lineage-specific structural adaptation in which NDH-II enzymes depend on an HMP scaffold, expanding their functional diversity beyond the classical monotopic paradigm.

Contrôle et régulation des voies de signalisation cellulaires: Les interactions protéine-protéine sont au centre de la signalisation cellulaire. Les MAPKs (Mitogen-Activated Protein Kinases) sont une famille de protéines kinases intracellulaires qui forment des cascades de signalisation contrôlant des mécanismes cellulaires fondamentaux tels que la prolifération, la différenciation, l'inflammation et la mort cellulaire. Les signaux progressent à travers différentes kinases (MAP4K, MAP3K et MAP2K) qui finissent par activer une MAPK. L'activation des MAPKs se fait par la double phosphorylation d'un motif TxY situé dans sa boucle d'activation, et module l'expression de gènes par l'intermédiaire de facteurs de transcription ou d'autres protéines kinases. La spécificité de liaison des MAP2Ks aux MAPKs est médiée par des motifs nommés KIM (pour Kinase Interacting Motif). Il existe de nombreuses structures aux rayons X des kinases individuelles et en complexe avec des KIMs isolés. Cependant, en raison de la nature transitoire de l'interaction entre les kinases, les détails moléculaires de la sélectivité et de l'activation d'une MAPK par sa MAP2K sont inconnus.Deux membres importants de la famille MAPK impliqués dans la réponse inflammatoire sont p38α, une MAPK, et sa MAP2K activatrice, MKK6. A travers eux, nous présentons le premier modèle de l'interaction entre une MAPK et sa MAP2K activatrice amont piégée à l'état de transition. Le modèle révèle un dimère de kinases face à face permettant à la boucle d'activation de p38α d'accéder au site actif de MKK6. La plupart des interactions sont éloignées par rapport aux sites de phosphorylation. En plus de l'interaction bien caractérisée entre le KIM de la MAPK2K et le "docking site" de la MAPK, une nouvelle interface d'interaction entre les lobes C des deux kinases a été indentifiée. Cette configuration jusqu'alors inobservée est favorable à la catalyse et, malgré sa résolution limitée, le modèle cryoEM de MKK6 et p38α ouvre la voie à des hypothèses sur l'activation des MAPK.Nous avons également découvert un nouveau mécanisme de phosphorylation non canonique de p38α par MKK6 utilisant l'ADP comme source de phosphate. Nous montrons que MKK6 est capable de phosphoryler p38α en utilisant le β-phosphate de l'ADP et de l'ATP in vitro, avec une efficacité moindre qu'avec l'hydrolyse canonique du γ-phosphate de l'ATP. Cette voie alternative de phosphorylation pourrait conférer un avantage adaptatif au stress en cas de privation d'ATP pour maintenir la voie de signalisation.Certains pathogènes détournent les voies de signalisation de leur hôte pour remodeler la réponse cellulaire à leur avantage. Le parasite intracellulaire Toxoplasma gondii sécrète des protéines effectrices qui imitent des motifs présents dans certaines protéines de l'hôte pour agir directement sur leurs cibles. Nous avons caractérisé comment deux effecteurs de T. gondii, GRA24 et GRA16, interagissent avec leur protéine cible.GRA24 se lie aux MAPKs de l’hôte p38α et ERK1/2 par le biais d'un motif KIM ; et GRA16 interagit avec la déubiquitinase hôte USP7 par le biais d'un motif de liaison MATH. Les deux motifs ont une haute affinité pour leur cible et se lient à des sites d’interaction connus en compétition avec les interacteurs de l'hôte. Nous présentons les détails moléculaires de l'interaction entre ces motifs pathogènes et leur cible et proposons un mécanisme d'action potentiel.

Highly pathogenic avian influenza viruses (HPAIVs) derive from H5 and H7 low pathogenic avian influenza viruses (LPAIVs). Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for the LPAIV-to-HPAIV transition, the mechanisms underlying the occurrence of insertion are unknown. Here, we show that transient H5 RNA structures, predicted to trap the influenza virus polymerase on purine-rich sequences, drive nucleotide insertions, providing empirical evidence of RNA structure involvement in MBCS acquisition. Introduction of H5-like sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions. Our results show that nucleotide insertions that underlie H5 HPAIV emergence result from an RNA structure–driven diversity-generating mechanism, which could also occur in other RNA viruses.

The RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway relays extracellular signals into a cellular response and its dysregulation leads to many pathologies, particularly cancer. Much is known about the individual components of the pathway, however, details of their interactions and how specificity is maintained is still poorly understood. Here, we determined cryo-EM structures of the MAP2K MEK1 activating its substrate MAPK ERK2, the final event in the cascade. We define the molecular details of specificity and activation of ERK2 and examine the mechanism of substrate recognition using solution techniques and molecular dynamics. Binding of the substrate MAPK leads to release of the MAP2K catalytic machinery, a mechanism enhanced in many gain of function disease mutations. Further, we observe that ERK2 release is not required for nucleotide exchange, suggesting how a processive mechanism could proceed. Our data advance the understanding of MAPK signalling specificity and provide a starting point for drug development. One-Sentence Summary: Cryo-EM structures of the MEK1-ERK2 complex reveal details of cellular signal transmission.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Phosphorylation is a fundamental mechanism in eukaryotic cells that allows signals to propagate. Kinases orchestrate this process by phosphorylating proteins to modulate their activity. Our work reveals the architecture of the complex between two key players of the mitogen-activated protein kinase signaling pathway, p38α and MKK6, while also giving an idea of what happens when the two come together.

Summary: Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein group across all kingdoms of life, making up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyse these reactions is essential in characterising the processes they regulate. Metal fluorides can be used as multifunctional tools for the study of these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogues. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryoEM and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal with soft X-rays. Highlights: ⍰ Enzymes that catalyse phosphoryl transfer are the largest family of enzymes and are involved in the storage and transmission of genetic information, energy transfer, signalling and cellular differentiation ⍰ Metal fluorides form a comprehensive tool kit to study the mechanisms of these enzymes by stabilizing the active conformation; mimicking both the transition state and ground state; and placing spectroscopically sensitive atoms into the active site ⍰ A guide is presented to the optimal formation of these complexes and their use in a wide variety of techniques in structural biology

Summary: Kinases are a diverse group of enzymes that use ATP to phosphorylate a variety of substrates. Protein kinases evolved in eukaryotes as important mediators of cell signalling that target specific amino acid side chains to modulate downstream protein function. Among them, the MAPKs (mitogen-activated protein kinases) are a family of intracellular protein kinases that form signalling cascades responding to a number of stimuli, that control fundamental mechanisms such as proliferation, differentiation, inflammation and cell death. Signals propagate through consecutive kinases which eventually phosphorylate and activate a MAPK. Here, we show that the dual specificity threonine/tyrosine MAP kinase kinases (MAP2Ks or MEKs) are able to phosphorylate and activate their substrate MAPKs using ADP as well as ATP in vitro . As the pathways are involved in the stress response, we speculate that it would represent an advantage to be able to maintain signalling under conditions such as hypoxia, that occur under a number of cell stresses, including cancer and atherosclerosis, where the available pool of ATP could be depleted.

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo–electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.