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      Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

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          Abstract

          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.

          Editor’s summary

          Mitogen-activated protein kinases (MAPKs) are a key player in cellular responses to various stimuli in eukaryotes. Signaling cascades occur through a series of upstream kinases, eventually resulting in double phosphorylation of MAPK that occurs in a complex that is transient and dynamic and thus difficult to visualize by traditional structural approaches. Juyoux et al . combined cryo–electron microscopy, biophysical techniques, and molecular dynamics simulations to construct a model of the active complex between the MAPK p38α and its upstream kinase, MKK6. Based on this model, the authors discuss specific interactions, selectivity, and the overall mechanism of p38α activation. These findings will be important for researchers seeking to target MAPKs for drug development. —Michael A. Funk

          Abstract

          Multimodal structural and modeling analyses are applied to an active hetero-kinase complex.

          Abstract

          INTRODUCTION

          Mitogen-activated protein kinases (MAPKs) are important signaling proteins found in eukaryotes. They respond to external signals and regulate various cellular processes. One such MAPK is p38α, which plays a crucial role in cell stress, inflammation, and response to infection, including the cytokine storm associated with severe COVID-19 and influenza. Dysregulation of p38α signaling is linked to several diseases, making p38α an important drug target. Understanding the interactions between p38α and its upstream activating MAP2K, MKK6, is essential to devise drug development strategies targeting allosteric sites. The MAP2Ks have a kinase interaction motif (KIM, or D motif) at the beginning of their intrinsically disordered N termini, which interacts with an allosteric docking site on their target MAPK, ensuring some level of specificity. Once engaged, the MAP2K phosphorylates a TxY motif (where T is threonine, Y is tyrosine, and x is any residue) on the MAPK activation loop (A loop), allowing it to adopt an active conformation. Whereas the individual kinases and their interactions through KIM motifs have been extensively studied, the overall interactions between components of the MAP kinase cascades are not yet fully understood. In particular, how specificity is maintained between different MAPK pathways and how MAP2Ks can phosphorylate both tyrosine and threonine residues—which is highly unusual in protein kinases—is unknown.

          RATIONALE

          The interaction between the kinases needs to be transient in order to maintain signal transmission. However, this fast interaction time hinders structural studies. We used a multidisciplinary approach—including cryo–electron microscopy (cryo-EM), small-angle x-ray scattering (SAXS), enhanced sampling molecular dynamics (MD) simulations, Bayesian modeling, hydrogen-deuterium exchange mass spectrometry, and cellular assays—to characterize the complex between p38α (MAPK14) and its activating MAP2K, MKK6 (MAP2K6).

          RESULTS

          The study provides a detailed molecular model of the dynamic MKK6-p38α complex, shedding light on specificity and multistep catalysis within the MAP kinase pathway. The cryo-EM structure shows that the kinases adopt a face-to-face conformation, with all contact between the kinases distal to the MKK6 active site. The structure reveals two major contacts occurring during complex formation: The MKK6 KIM binds at the expected p38α common docking site, and the MKK6 αG helix engages the p38α C lobe at the so-called lipid-binding site that is specific to MAPKs and has been implicated in regulation of the pathway. MD simulations reveal that the observed conformation facilitates the approach of the A loop of p38α to the active site of MKK6 without compromising the dual specificity of MKK6. The simulations show that both the A-loop threonine and tyrosine can access the active site without specific recognition or binding of the A loop. Adaptive-sampling MD simulations combined with solution SAXS data in a Bayesian/maximum-entropy approach were then used to reconstruct the heterogeneous conformational ensemble and understand how the two kinases assemble and initiate phosphorylation. The populations of the main states captured in this ensemble, as well as the reaction paths connecting them, show the importance of the N terminus of MKK6 and the C lobes of the two kinases in correctly positioning them for phosphorylation. Cellular assays performed with variants of MKK6 N termini revealed that the length and structure of the N-terminal linker are important in determining specificity between MAP2K and MAPK pairs.

          CONCLUSION

          Resolving the architecture of a MAP2K activating its target MAPK has identified the interaction sites between the two kinases and allowed us to model the mechanism of activation and discover components that are important in specificity. The N termini of MAP2Ks guide the engagement of specific kinases by being tuned to the correct distance for MAP2K/MAPK pairs. Once bound, rather than acting like a classical enzyme and positioning substrates precisely for catalysis, MKK6 creates a zone of proximity enabling either the tyrosine or threonine to approach the active site, regardless of their state, allowing dual specificity. Through a comprehensive multidisciplinary approach, the study elucidated the architecture and dynamics of the formation of the MKK6-p38α complex. The findings pave the way for targeted drug development and enhance our understanding of essential steps in kinase signaling cascades.

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          Most cited references111

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            SWISS-MODEL: homology modelling of protein structures and complexes

            Abstract Homology modelling has matured into an important technique in structural biology, significantly contributing to narrowing the gap between known protein sequences and experimentally determined structures. Fully automated workflows and servers simplify and streamline the homology modelling process, also allowing users without a specific computational expertise to generate reliable protein models and have easy access to modelling results, their visualization and interpretation. Here, we present an update to the SWISS-MODEL server, which pioneered the field of automated modelling 25 years ago and been continuously further developed. Recently, its functionality has been extended to the modelling of homo- and heteromeric complexes. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex are inferred by homology modelling. Other major improvements include the implementation of a new modelling engine, ProMod3 and the introduction a new local model quality estimation method, QMEANDisCo. SWISS-MODEL is freely available at https://swissmodel.expasy.org.
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              cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

              A software tool, cryoSPARC, addresses the speed bottleneck in cryo-EM image processing, enabling automated macromolecular structure determination in hours on a desktop computer without requiring a starting model.
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                Author and article information

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                Journal
                Science
                Science
                American Association for the Advancement of Science (AAAS)
                0036-8075
                1095-9203
                September 15 2023
                September 15 2023
                : 381
                : 6663
                : 1217-1225
                Affiliations
                [1 ]European Molecular Biology Laboratory (EMBL), Grenoble, France.
                [2 ]Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland.
                [3 ]School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.
                [4 ]European Synchrotron Radiation Facility, Grenoble, France.
                [5 ]Protein and peptide purification platform, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
                [6 ]Department of Chemistry, University College London, London, UK.
                [7 ]Institute of Structural and Molecular Biology, University College London, London, UK.
                [8 ]Swiss Institute of Bioinformatics, Geneva, Switzerland.
                Article
                10.1126/science.add7859
                37708276
                26cf50aa-27df-435b-89c0-e823bf99a8da
                © 2023

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