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Understanding how the cytokine storm propagates

When cells are put under stress from their environment they need to react. To fight stress, such as infection, several coping mechanisms, including the inflammatory response, are used by our bodies. While inflammation is necessary, too much of it can impair cell and organ function. This is the case with cytokine storms – inflammatory cascades during an infection that can spiral out of control and lead to severe disease and even death, as recently highlighted during the COVID-19 pandemic.

When a cell receives the message to start inflammation, the signal is relayed through a series of protein kinases, called the Mitogen Activated Protein (MAP) kinases, that phosphorylate and activate one another. First a MAP kinase kinase kinase (MAP3K) activates a MAP kinase kinase (MAP2K) which in turn activates a specific MAPK. The final MAPK in the chain then enters the nucleus where it modulates gene transcription allowing the cell to react. The chain of kinases allows the signal to be amplified, as each activated kinase can itself activate many more of its target kinases. The individual components of this relay have been studied extensively over 30 years but as the interactions have to be transient in order to transmit the signal, how these proteins interact is largely unknown.

We studied the MAP kinase p38α responsible for activating the inflammatory response, in complex with its upstream activating MAP2K, MKK6 [1]. The key role of p38α in inflammation and the fact that aberrant p38α signaling is involved in numerous diseases, such as arthritis and cancer, but also in the response to infection, make it a highly studied drug target [2]. We used cryo-electron microscopy (cryoEM) to determine the structure of the complex at 4 Å resolution revealing a ‘face-to-face’ conformation of the kinases with the activation loop (A-loop) of p38α, the region phosphorylated by MKK6, extending towards the active site (Figure 1). The structure reveals new interactions between the kinases and, intriguingly, all contact between the kinases is distal to the MKK6 active site. In order to further our understanding of the system we turned to molecular dynamics simulations, revealing 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. Extending these simulations to much longer timescales using a Bayesian/maximum-entropy approach and refinement against SAXS data allowed us to reconstruct the heterogeneous conformational ensemble and understand how the two kinases assemble and initiate phosphorylation (Figure 2). The populations of the main states captured in this ensemble, as well as the 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 kinase pairs.

Resolving the architecture of MKK6 activating its target MAPK p38α has identified previously unknown interaction sites between the two kinases and allowed us to model the mechanism of activation. 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. Many PSB platforms contributed to this work including the EMBL EEF, the macromolecular crystallography and SAXS beamlines of the ESRF-EMBL JSBG and mass spectrometry at the IBS. The findings pave the way for targeted drug development and increase our understanding of essential kinase signaling cascades.

M.W. Bowler (EMBL), E. Pellegrini (EMBL, now IBS) and P. Juyoux (EMBL, now IBS)

[1] P. Juyoux, I. Galdadas, D. Gobbo, J. von Velsen et al. (2023). Science 381, 1217-1225.

[2] B. Canovas and A. R. Nebreda (2021) Nat. Rev. Mol. Cell Biol. 22, 346–366.

Figure caption

Figure 1: Structure of the MKK6-p38α complex.

Figure 2: Population of states during the assembly of p38α and MKK6 as derived from the fitting of MD states to the SAXS curve.