title: A conserved isoleucine maintains the inactive state of Bruton's tyrosine kinase.
abstract: Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a "wedge" that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Boyken
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Nikita
authors.initials: N
authors.lastname: Chopra
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Raji E
authors.initials: RE
authors.lastname: Joseph
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: D Bruce
authors.initials: DB
authors.lastname: Fulton
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Jernigan
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA. Electronic address: amyand@iastate.edu.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Andreotti
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D007532:Isoleucine / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D018909:src Homology Domains / Q000235:genetics
chemical_list: D007532:Isoleucine; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; D007930:Leucine
keywords: Tec kinase regulation; active/inactive conformational equilibrium; catalytic activity; isoleucine/leucine; molecular dynamics