Computational and Experimental Characterization of Intramolecular Regulatory Interactions in Hck

Abstract

Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases that play critical roles in cellular transduction pathways. These highly sought after therapeutic targets are prevalent and promiscuous; therefore, they must be tightly regulated to prevent unchecked signaling cascades. Inter-domain interactions that hinder kinase function are key SFK regulatory mechanisms, and detailed information about these interactions is integral to understanding how these enzymes function.Hck is a SFK primarily found in cells of hematopoietic lineage and is among the most well studied members of the family. Crystal structures of Hck in the down-regulated form and solution studies detailing catalytic responses to regulatory domain displacement provide a robust framework for characterizing the regulatory interactions. Adding to this knowledge, we provide fundamental information on Hck regulation by measuring binding free energies of the native domain-peptide interactions. A combined experimental and computational approach was used to complement and expand the usefulness of the data. The results were compared with bulk FRET measurements made using the full-length kinase to observe how these interactions are modified in the context of the protein.Given the high degree of homology among family members, it is unsurprising that SFKs have some redundant or compensatory functions. However, it has been shown that even the most closely related members of the family cannot always substitute functionally for one another in vivo and the differences responsible have yet to be fully elucidated. Our results lay the groundwork for comparative analysis between different family members and are expected to aid in identifying features that distinguish these enzymes from one another.