Crystal Structures of the Phosphorylated BRI1 Kinase Domain and Implications for Brassinosteroid Signal Initiation

Let’s get started with my very first scientific publication! Back in the day during my undergraduate (doing my BSc in Biochemistry at the Eberhard-Karls-University in Tuebingen) I was working in the laboratory of Dr. Michael Hothorn (who is now a professor at the University of Geneva) at the Friedrich-Miescher-Laboratory of the Max-Planck-Society. Michael is interested in plant signaling and uses a mix of structural biology and biochemistry to dissect these pathways.

Wanting to learn more about the structure of proteins and signaling pathways, I started to work on the cytoplasmic kinase domain (an enzymatic domain adding phosphates to other protein) of the plant brassinosteroid receptor BRI1 (brassinosteroid insensitive 1), which senses these plant steroids regulating size and shape of the plant. While steroid receptors in animals reside in the cell nucleus, steroid receptors in plants are transmembrane receptors. BRI1 specifically signals together with its co-receptor BAK1 (BRI1-associated kinase 1) after it binds brassinolide.

One reason for interest in the structure of the kinase domain of BRI1 is that BRI1 is a dual-specificity kinase, meaning that it can phosphorylate the amino acids serine and threonine as well as tyrosine, an unusual feat for a kinase. To rationalize this functionality, a precise three-dimensional structure of BRI1’s kinase domain would be needed, which we intended to do with X-ray crystallography. For this, ordered crystals of the kinase domain are bombarded with high-energy X-rays and the three-dimensional coordinates of the protein can be reconstructed from the diffracted X-rays. But first, sufficient amounts of the protein had to be produced.

Overproducing the kinase domain (or more precisely several variants of it as it was unclear which one would successfully crystallize) in commonly used Escherichia coli cells allowed to then extract the proteins and purify them with various chromatographic methods (to be technical: two His-tag, one anion exchange and one size exclusion chromatography). Having arrived at pure proteins of uniform size and charge, I then tried to coax these proteins into crystallizing by trying to mix them with hundreds of different chemical formulations known to enable protein crystallization.

Initially, this proved to be a difficult task, as no successful condition was found. Only after mutating the kinase domain into a hyperactive version (to ensure a uniform autophosphorylation pattern), cutting off flexible parts and reductively methylating its surface lysines (to further decrease the flexibility) did I find conditions which facilitated crystallization. I then optimized these conditions and solved several structures of the kinase domain. Equipping the protein with diverse stages of its substrate ATP (adenosine triphosphate; enabling the kinase domain to phosphorylate other proteins) led to structures in different stages of activation.

By investigating the structure of the BRI1 kinase domain, we found a structural similarity to the animal Pelle/IRAK kinase family, which is also known to harbor dual-specificity kinases. Additionally, together with data from analytical size exclusion chromatography and western blots, we could identify the C-terminal lobe of the BRI1 kinase domain as an important interaction point with its co-receptor BAK1.

Next to teaching me copious amounts of methods and experimental knowledge, this project also furthered our knowledge of the signaling initiation process upon the binding of brassinosteroids in plants. Fortunately, we were able to publish this work in the Plant Journal and I still keep a 3D-printed rendering of the BRI1 kinase domain as a memento on my desk.

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