Inflammation is part of the body's normal response to infection and other stresses. In eukaryotes, mitogen-activated protein kinases (MAPKs) form signalling cascades that respond to extracellular stimuli and transmit a signal through the cell membrane and down into the nucleus, activating the genes for the inflammatory response. Although MAPKs have been extensively studied, their rapid interactions during the signalling cascades are challenging to explore. In work recently published in Science, an international team of researchers used a multidisciplinary approach - including cryo-electron microscopy (cryo-EM), small-angle X-ray scattering (SAXS), enhanced sampling molecular dynamics (MD) simulations, Bayesian modelling, hydrogen-deuterium exchange mass spectrometry, and cellular assays to investigate how MAPK p38ɑ, the final switch regulating inflammation, is activated by its upstream kinase. Their study captures a fundamental step of cell signalling, enhances our understanding of essential steps in kinase signalling cascades and paves the way for the targeted development of new therapies to stop cytokine storms.
During the first stages of the Covid-19 pandemic, the general public became familiar with the concept of a 'cytokine storm', a situation in which the immune system goes into overdrive, getting sidetracked from fighting an infection and instead attacking the body itself. A range of conditions can trigger a cytokine storm, and researchers are working on new ways to diagnose and treat them.
The release of cytokines, pro-inflammatory signalling molecules, is initiated by a cascade of kinases. One of these kinases, p38ɑ, has a key role in the signalling cascade and is linked to several diseases, which makes it an important drug target.
Dr Matthew Bowler, a researcher at EMBL Grenoble who has been studying kinases for over a decade says;
p38ɑ is interesting because it's responsible for starting inflammation from lots of different places, so a drug targeting p38ɑ would broadly target inflammation. The structure of it has been known for 25 years, but the main drug target is the nucleotide binding site, which is similar in all kinases, and that lack of specificity leads to a lot of off-target effects. Although many molecules that have been designed to target p38α, none have yet made it past clinical trials due to this lack of specificity.
The information structural biologists have on MAPKs has mainly been gathered in isolation, ignoring how the enzymes interact during the cascade. Developing a better understanding of the molecular dynamics is challenging, due to the transient nature of the interactions.
Dr Bowler continued;
We wanted to understand the interactions between p38ɑ and the kinase that activates it (MKK6). Transmitting the signal involves a very fast interaction between these two proteins, which then separate again. Using SAXS on Diamond's B21 beamline allowed us to capture that brief moment of interaction. We tried various methods of stabilising the two kinases together, and the SAXS data we collected showed us which of those methods was most successful. Once we had the stabilised kinases, we were able to use cryo-EM to solve the structure.
The 3D structure of the complex (Fig. 1) allowed the team to identify a potential new druggable pocket - a previously unknown docking site where the two enzymes interact. Feeding the structure into molecular dynamics simulations, in collaboration with the group of Prof. Francesco Gervasio in Geneva University, offered further insights into how the two kinases interact (Fig. 2).
The SAXS data were also a vital component of the molecular dynamics simulations, as Dr Bowler explained:
With the cryo-EM structure we have a snapshot of the two kinases interacting, but the molecular dynamics look at a much longer timescale, and give us an idea of what's happening with these two proteins before you can lock them together when they're allowed to move around freely. And the SAXS data were invaluable here, too, because it shows all the different states in solution, and reveals the mechanism of the formation of the complex.
In this research, the team were investigating one particular MAPK pathway, which is for inflammation. Their work has highlighted a lot of potential drug targets, offering the hope that new therapies will one day be able to prevent damaging cytokine storms. The team is now using the same approach to investigate other MAPK pathways, which could ultimately lead to treatments for other diseases, such as arthritis, Alzheimer's and cancer.
To find out more about the B21 beamline or discuss potential applications, please contact Principal Beamline Scientist Nathan Cowieson: firstname.lastname@example.org.
Juyoux P et al. Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation. Science 381.6663: 1217-1225 (2023). DOI:10.1126/science.add7859.
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