Protein phosphorylation is an important cellular regulatory mechanism which acts like a molecular switch in our cells. It is a process where a phosphate group, a small but very consequential chemical tag, is added to a protein. Think of it as turning a light switch on or off: when a protein gets tagged with a phosphate group, it's often turned "on" or activated. Phosphorylation is one of the main strategies cells use to propagate the signals they receive from their environment and control the activity of proteins. This strategy allows a quick adaptation to changes in the environment - a vital reaction for various functions in your body. 

Signal propagation through phosphorylation typically happens in ‘cascades’ of cellular events, similar to a relay race where a message is passed from one runner to another. Among the many runners within a cell, protein kinases transmit signals by adding a phosphate group on their target protein, often another kinase, propagating the signal until ultimately triggering a specific response in the cell. The kinases MKK6 and its substrate p38α, both Mitogen-Activated Protein Kinases (MAPK), play a crucial role in transmitting signals related to cell stress and inflammation. 

Like in the relay race, the interaction between the kinases needs to be transient to allow the phosphorylation of multiple target proteins sequentially and signal amplification. However, this fast interaction time hinders structural studies that could allow us to see what happens when two kinases come together to phosphorylate one another and give us insights as to how we could interfere with this interaction associated with human diseases. 

Therefore, we asked whether we could engineer the complex to stabilise the highly transient p38α -MKK6 dimer in its active form. We introduced the equivalent sequence from the Toxoplasma gondii protein GRA24 at the beginning of the MKK6 in a region called the kinase interaction motif, known to interact with p38α. Thanks to this engineering, we were able to increase the affinity of MKK6 for p38α and stabilise the complex. We also inserted two mutations in the MKK6 activation loop to transform the kinase into an active form that is ready to phosphorylate p38α. 

Then, using cryo-electron microscopy (cryo-EM) - a technique used to visualise the three-dimensional structures of biological molecules at high resolution by rapidly freezing them and capturing images of their electron-scattering patterns - we resolved the structure of p38α in complex with MKK6 in a pre-phosphorylation state. The cryo-EM structure showed that the kinases adopt surprisingly a face-to-face conformation with all contacts between the kinases far from the MKK6 active site. 

Since this conformation had never been described before, we had to be sure that the observed face-to-face complex corresponds to the ‘real’ complex involved in the physiological phosphorylation mechanism. To ensure that our engineered complex does mimic the endogenous protein in our cells, we compared the behaviour of the mutant and wild-type complexes thanks to a combination of two methods: the Hydrogen/Deuterium exchange coupled with Mass Spectrometry technique monitors protein dynamics and allows the mapping of protein interaction sites, while molecular dynamics simulations, a computer simulation technique, lets us follow the behaviour of the complex over time. Both methods showed that the contacts between the two proteins were the same in the engineered and wild-type complex. 

 

The simulations also revealed that the conformation we see in the cryo-EM facilitates the approach of the activation loop of p38α to the active site of MKK6 without compromising MKK6’s ability to phosphorylate the two known sites on p38α. What is more, we were able to observe the mechanism by which the two kinases approach one another and the different conformations that they adopt to initiate phosphorylation. 

Finally, by performing a series of cellular assays using variants of MKK6, we found that the length and structure of the kinase interaction motif linker are essential in making MKK6 specific to p38α. 

Characterising the architecture of MKK6 activating its target p38α not only opens exciting new paths to better understand the complex’s inner workings, but also provides crucial information to design new drugs that can modulate the inflammation response regulated by p38α.