Biochem J. 2009 Apr 28;420(1):115-22. doi: 10.1042/BJ20081405.

Identification of dimer interactions required for the catalytic activity of the TRPM7 alpha-kinase domain.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Crawley, S. W., Cote, G. P.,
["Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6."]
TRPM7 (transient receptor potential melastatin) combines an ion channel domain with a C-terminal protein kinase domain that belongs to the atypical alpha-kinase family. The TRPM7 alpha-kinase domain assembles into a dimer through the exchange of an N-terminal segment that extends from residue 1551 to residue 1577 [Yamaguchi, Matsushita, Nairn and Kuriyan (2001) Mol. Cell 7, 1047-1057]. Here, we show, by analysis of truncation mutants, that residues 1553-1562 of the N-terminus are essential for kinase activity but not dimer formation. Within this 'activation sequence', site-directed mutagenesis identified Tyr-1553 and Arg-1558 as residues critical for activity. Examination of the TRPM7 kinase domain structure suggests that the activation sequence interacts with the other subunit to help position a catalytic loop that contains the invariant Asp-1765 residue. Residues 1563-1570 of the N-terminal segment are critical for dimer assembly. Mutation of Leu-1564, Ile-1568 or Phe-1570 to alanine abolished both kinase activity and dimer formation. The activity of a monomeric TRPM7 kinase domain lacking the entire N-terminal segment was rescued by a GST (glutathione transferase) fusion protein containing residues 1548-1576 of TRPM7, showing that all interactions essential for activity are provided by the N-terminal segment. Activity was also restored by GST fused to the N-terminal segment of TRPM6 (residues 1711-1740), demonstrating the feasibility of forming functional TRPM6-TRPM7 alpha-kinase domain heterodimers. It is proposed that covalent modifications or binding interactions that alter the conformation of the N-terminal exchanged segment may provide a means to regulate TRPM7 kinase activity.
PMID: 19228120External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TRPM7 Fusion protein-pull down assay Mouse 1711-2028 Mouse 1548-1863 19228120
TRPM7 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TRPM6 Fusion protein-pull down assay Mouse 1548-1863 Mouse 1711-2028 19228120
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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