Taking a chemical biology approach: Semisynthesis of the EPH receptor tyrosine kinase for structural and mechanistic studies.

摘要

The Eph receptors are the largest subfamily of receptor tyrosine kinases and along with their membrane bound ephrin ligands set up unique bidirectional-signaling pathways. However, to date there is no information on how extracellular ligand binding is translated, in a series of molecular steps, into activation of the intracellular kinase domain in any Eph receptor, or for that matter, any receptor kinase. As for most membrane proteins, the main impediment is the lack of expression systems to produce high amounts of purified, biologically active Eph molecules. Our work is aimed at overcoming this problem by using a novel chemical biology approach, namely expressing the functional extracellular and intracellular domains separately using different expression systems, and then linking them together in vitro using a chemical ligation reaction. Hence, we have further developed the semi-synthetic technique of expressed protein ligation (EPL) to reconstitute the complete Eph receptor for structural and mechanistic studies. EPL is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. We have demonstrated for the first time that using the EPL technique, the multi-domain, disulfide bonded extracellular region of the Eph receptor can be modified with synthetic peptides, while its biological activity is retained. We have extended this work to ligating the complete ectodomain of the Eph receptor to the intracellular juxtamembrane and tyrosine kinase domains. We document that all functional Eph receptor domains fully retain their biological activity. The semisynthetic receptors can be produced in milligram amounts, are highly pure and monodisperse, and are thus ideal for structural and functional analysis Importantly, stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phospho-proteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. We are using X-ray crystallography and cryoelectron microscopy for structural characterization of this semisynthetic construct. We have also made significant progress in incorporating the residues corresponding to the Eph transmembrane region in our current construct, which will allow for studying its precise role in the receptor activation mechanism. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.