Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism

Abstract

Summary The DAN-family, including Gremlin-1 and Gremlin-2 (Grem1 and Grem2), represents a large family of secreted BMP antagonists. However, how DAN proteins specifically inhibit BMP signaling has remained elusive. Here, we report the structure of Grem2 bound to GDF5 at 2.9 Å resolution. The structure reveals two Grem2 dimers binding perpendicularly to each GDF5 monomer, resembling an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N-terminus that undergoes significant transition upon complex formation, leading to simultaneous interaction with the type I/type II receptor motifs on GDF5. Binding studies show that DAN-family members can interact with BMP-type I receptor complexes, whereas Noggin outcompetes the type I receptor for ligand binding. Interestingly, Grem2-GDF5 forms a stable aggregate-like structure, in vitro, not clearly observed for other antagonists, including Noggin and Follistatin. These findings exemplify the structural and functional diversity across the various BMP antagonist families.

Analysis and identification of the Grem2 heparin/heparan sulfate-binding motif

“…Despite these minor observed differences, we believe that these data suggest that mutation of these specific lysine residues does not significantly alter the ability of Grem2 to bind to or inhibit BMP2 signaling, suggesting that the heparin/HS-binding motif and the BMP-binding motif are probably distinct in nature. Furthermore, this is consistent with the recent structure of Grem2–GDF5, where the lysine residues mutated in the present study are not located within or near the observed Grem2–ligand interface, thus appearing to play no role in direct BMP binding or inhibition [25]. …”

“…Similar to Grem2, BMP2 maintains the ability to bind heparin/HS using amino acids located within the N-terminus of the ligand, losing this ability upon removal of these basic residues [33]. Using our recently published Grem2–GDF5 structure, we generated a working model of the Grem2–BMP2 complex [25]. In this model, similar to our published structure, two molecules of Grem2 can be found binding to the distal ends of the ligand dimer (Figure 6A).…”

“…Surface plasmon resonance (SPR) experiments were performed at room temperature using a Biacore T200 as previously described [24,25]. Recombinant mature BMP2 was purified as previously described and diluted in sodium acetate buffer (pH 4.5) prior to immobilization on a CM5 sensor chip using the standard EDC/NHS primary amine coupling chemistry and according to the manufacturer’s protocol [25,26].…”

“…Recombinant mature BMP2 was purified as previously described and diluted in sodium acetate buffer (pH 4.5) prior to immobilization on a CM5 sensor chip using the standard EDC/NHS primary amine coupling chemistry and according to the manufacturer’s protocol [25,26]. To determine the binding kinetics of Grem2 WT and mutants, serial dilutions in HBS–EP buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% v/v P20 surfactant] were injected at a constant flow rate of 30 μl/min for an association of 3 min and allowed to dissociate for 10 min.…”

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

“…Specifically, GREM2 dimerizes in a head-to-tail manner, unlike the head-to-head pairing of NOGGIN [24,46,47]. This head-to-tail arrangement gives rise to large, constrained, and arching hydrophobic surfaces on the threedimensional structure, which precludes Grem2 from wrapping around BMP dimers as NOGGIN does [24,46,47]. We currently test whether this unique structural arrangement is also critical for the function of GREM2 in regulating both proliferation and differentiation of CPCs.…”