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Competing modes of self-association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization

¹ Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50010, USA
² Department of Chemical Engineering, Iowa State University, Ames, Iowa 50010, USA
³ Program of Bioinformatics and Computational Biology, Iowa State University, Ames, Iowa 50010, USA
⁴ Department of Physiology, Cornell University Medical College, New York, New York 10021, USA

Reprint requests to: Dr. Amy H. Andreotti, Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50010; e-mail: amyand{at}iastate.edu; fax: (515) 294-0453.

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.

Keywords: Bruton's tyrosine kinase (Btk); SH3; NMR; asymmetric homodimer; intramolecular; self-association

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