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S C I E N T I F I C H I G H L I G H T S

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Fig. 21: Structure of the MKK6-p38α complex.

Understanding how the cytokine storm propagates

A combination of X-ray scattering, cryo-electron microscopy experiments and theoretical calculations have revealed the architecture of the complex between two key players of the mitogen-activated protein kinase signalling pathway, p38α, and its activating kinase, MKK6, which triggers inflammation during infection.

Cell signalling is an essential mechanism to allow organisms to react to their environment. When cells are put under stress from their environment they need to react. To fight stress, such as infection, cells have developed several coping mechanisms, including the inflammatory response. 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.

This work studied the MAP kinase p38α, which is responsible for inflammation, in complex with its upstream activating MAP2K, MKK6. Using small-angle X-ray scattering (SAXS) at beamline BM29, and at Diamond Light Source (UK) beamline B21, various mutants were studied and the most stable combination of the kinases found. Cryo-electron microscopy (cryo-EM) was used 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 21). As the resolution of the cryo-EM data was limited, the high-resolution structures of p38α were determined at beamlines ID23-1 and ID23-2 in order to have a good model to place in the cryo-EM density. X-ray centring procedures [1] and micro-focused beams were used to gain the best possible data due to the small size and variability of the crystals. These structures were then refined against the cryo-EM map.

The structure reveals new interactions between the kinases and, intriguingly, all contact between the kinases is distal to the MKK6 active site, which probably allows flexibility in the residue that the kinase can target. Molecular dynamics simulations revealed 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.