SH2-Catalytic Domain Linker Heterogeneity Influences Allosteric Coupling across the SFK Family

Abstract

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators

“…Linkers play a variety of structural and functional roles in naturally occurring proteins. For example, they have a role in tuning of biological activities of the connected domains [3] , [4] , in allosteric coupling [5] , in viral replication [6] . They are also of the utmost interest and relevance for applications in protein engineering, for example the alteration of functionality of engineered antibodies [7] , [8] , [9] .…”

Paralog-Specific Patterns of Structural Disorder and Phosphorylation in the Vertebrate SH3–SH2–Tyrosine Kinase Protein Family

“…CSK orchestrates the inhibition of SRC paralogs ( Okada 2012 ) which, in contrast, exert an activation effect on TEC paralogs ( Qiu and Kung 2000 ) and on ABL paralogs ( Colicelli 2010 ). Sequence divergence at regions such as the A-loop, determines the different catalytic efficiencies in TKs ( Joseph et al 2013 ), while sequence heterogeneity of linkers L3 play another important role regulating catalysis via allosteric coupling ( Register et al 2014 ). The differential disorder predicted between paralogs in the same family such as MATK-CSK and BLK-LCK agrees with reported noncatalytic activities for MATK ( Chong et al 2006 ) and LCK ( Rossy et al 2013 ) and their accumulated fractions of disorder after the gene duplications.…”

Dynamic regulatory features of the protein tyrosine kinases

“…Moreover, the nonconserved histidine at the bottom of the HCK hydrophobic stack may be in a protonated or deprotonated state (65) creating further potential deviations from the well aligned hydrophobic stack residues observed in SRC. Sequence differences in the SH2-kinase linker of the SRC family, in particular between splice variants of FYN, have also been tied to regulation of kinase activity (66,67) and it is intriguing to consider that hydrophobic stack assembly may respond to differences in linker sequence and dynamics.…”