title: Competing modes of self-association in the regulatory domains of Bruton's tyrosine kinase: intramolecular contact versus asymmetric homodimerization.
abstract: A nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer-dimer equilibria. The Btk fragment studied contains two consecutive proline-rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino-terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy-terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino-terminal proline stretch; the other, in the carboxy-terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino-terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined K(a) values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co-localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.
authors.affiliation: Department of Chemical Engineering, Iowa State University, Ames, Iowa 50010, USA.
authors.forename: Alain
authors.initials: A
authors.lastname: Laederach
authors.forename: Kendall W
authors.initials: KW
authors.lastname: Cradic
authors.forename: Kristine N
authors.initials: KN
authors.lastname: Brazin
authors.forename: Jamillah
authors.initials: J
authors.lastname: Zamoon
authors.forename: D Bruce
authors.initials: DB
authors.lastname: Fulton
authors.forename: Xin-Yun
authors.initials: XY
authors.lastname: Huang
authors.forename: Amy H
authors.initials: AH
authors.lastname: Andreotti
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
chemical_list: D011392:Proline; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D000409:Alanine