ALK2 Receptor Kinase Association with FKBP12.6 Is Structurally Conserved with the ALK2-FKBP12 Complex

Abstract

The immunophilin FKBP12 is a known inhibitor of type I BMP and TGF-β receptors that competes for binding with their substrate SMADs. FKBP12 and the close paralog FKBP12.6 additionally assemble with ryanodine receptors to control Ca²⁺ release. Binding of FKBP12.6 to BMP/TGF-β receptors has yet to be investigated, but appears plausible given its high sequence similarity to FKBP12. Here, we found that FKBP12.6 can assemble with BMP and TGF-β-family type I receptors, but not with type II receptors. Cellular immunoprecipitation confirmed similar binding of FKBP12 and FKBP12.6 to the BMP receptor ALK2 (ACVR1), a known target of mutations in the congenital syndrome fibrodysplasia ossificans progressiva (FOP), as well as the pediatric brain tumor diffuse intrinsic pontine glioma (DIPG). SEC-MALS analyses using purified proteins indicated a direct 1:1 interaction between FKBP12.6 and the receptor's cytoplasmic domains. The 2.17 Å structure of this ALK2-FKBP12.6 complex bound to the inhibitor dorsomorphin showed FKBP12.6 binding to the GS domain of ALK2 in a manner equivalent to the FKBP12 complex, with ALK2 residues Phe198 and Leu199 extending into the FK506-binding pocket of FKBP12.6. These findings suggest a level of redundancy in FKBP-family regulation of BMP and TGF-β signaling.

Keywords: ACVR1; ALK2; BMP; FKBP12; FKBP12.6; fibrodysplasia ossificans progressiva; kinase.

Figure 1

FKBP12.6 binds to type I receptors in a 1:1 complex. (A) Immunoprecipitation (IP) to assess ALK2 binding to FKBP12 or FKBP12.6 in MDA-MB-231 cells transfected with FLAG-tagged ALK2 and GFP-tagged FKBPs. Proteins were detected by Western blot (WB) using the indicated antibodies. (B) Analytical size exclusion chromatography elution profiles for the indicated individual recombinant proteins and upon mixing with FKBP12.6. (C) Multi-angle light scattering (MALS) data for the peak of the indicated elution samples.

Figure 2

Structure of the ALK2-FKBP12.6 complex with dorsomorphin. ALK2 (green) and FKBP12.6 (orange) are shown in ribbon representation with secondary structural elements labelled. Regions missing in the electron density maps, where the chain could not be modeled, are shown by dashed lines. The kinase inhibitor dorsomorphin bound in the ATP-binding site is shown in a stick representation with its carbon, oxygen, and nitrogen atoms displayed in yellow, red, and blue, respectively.

Figure 3

Interactions between ALK2 and FKBP12.6. (A) αGS2 residues ALK2 Phe198 and Leu199 (green) inserted into a hydrophobic pocket in FKBP12.6 (grey surface with hydrophobic patch shown in red). (B) Backbone hydrogen bond from ALK2 Leu199 to the side chain of FKBP12.6 Asp38. (C) Water-mediated hydrogen bond between ALK2 Arg202 and FKBP12.6 Gln54. (D) Hydrogen bonding between ALK2 Gln207 and FKBP12.6 Glu55 and Arg50.

Figure 4

Structural comparison of ALK2 binding to FKBP12 and FKBP12.6. Superposition reveals high structural conservation between the ALK2-FKBP12.6-dorsomorphin complex (green/orange/yellow) and the previously reported structure of the ALK2-FKBP12-dorsomorphin complex (purple/cyan/magenta)(PDB 3H9R [14]).

Figure 5

Structural variations between ALK2 binding to FKBP12 and FKBP12.6. Structures of ALK2-FKBP12 (PDB 3H9R [14]), ALK2-FKBP12.6 (PDB 4C02), and ALK5-FKBP12 (1B6C [12]) were superimposed and are displayed in the indicated colors. Different views of the FKBPs binding to the αGS2 helices of ALK2 and ALK5 are shown highlighting selected side chain and conformational differences. (A) FKBP12 Ile91 is replaced by Val91 in FKBP12.6, which packs against two distinct conformations of ALK2 Phe198. (B) FKBP12 Trp60 is replaced by Phe60 in FKBP12.6 and packs against ALK2 Leu199. (C) FKBP12.6 Glu55 and Arg50 can form an intramolecular interaction in addition to Glu55 interaction with ALK2 Gln207. (D) ALK2 Phe198-Leu199 are replaced by Leu195-Leu196 in ALK5. Both ALK5 leucines occupy the hydrophobic FK506-binding pocket in the FKBP12 co-structure (red). In the equivalent ALK2-FKBP12 structure, ALK2 Phe198 (magenta) folds outside this pocket. The ALK2-FKBP12.6 co-structure shows ALK2 Phe198 (green) adopting both conformations. (E) The conserved FKBP residue Tyr83 packs more closely to the αGS2 helix in the ALK2 co-structures compared to the ALK5 co-structure. (F) Sequence alignment of FKBP12 and FKBP12.6 highlighting conserved residues and secondary structural elements. Side chains displayed in the figure are denoted by an asterisk.