Structure of Gremlin-1 and analysis of its interaction with BMP-2

Abstract

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) signalling family and has a very broad biological role in development. Its signalling is regulated by many effectors: transmembrane proteins, membrane-attached proteins and soluble secreted antagonists such as Gremlin-1. Very little is known about the molecular mechanism by which Gremlin-1 and other DAN (differential screening-selected gene aberrative in neuroblastoma) family proteins inhibit BMP signalling. We analysed the interaction of Gremlin-1 with BMP-2 using a range of biophysical techniques, and used mutagenesis to map the binding site on BMP-2. We have also determined the crystal structure of Gremlin-1, revealing a similar conserved dimeric structure to that seen in other DAN family inhibitors. Measurements using biolayer interferometry (BLI) indicate that Gremlin-1 and BMP-2 can form larger complexes, beyond the expected 1:1 stoichiometry of dimers, forming oligomers that assemble in alternating fashion. These results suggest that inhibition of BMP-2 by Gremlin-1 occurs by a mechanism that is distinct from other known inhibitors such as Noggin and Chordin and we propose a novel model of BMP-2-Gremlin-1 interaction yet not seen among any BMP antagonists, and cannot rule out that several different oligomeric states could be found, depending on the concentration of the two proteins.

Keywords: Gremlin; X-ray crystallography; bone morphogenetic protein (BMP); differential screening-aberrative in neuroblastoma (DAN); extracellular antagonism; structural biology; transforming growth factor-β (TGF-β).

Figure 1. Preparation and functional analysis of Gremlin-1 constructs

(A) SDS-PAGE analysis of all Gremlin-1 constructs after final purification: untagged ΔN-Gremlin-1, His-tagged ΔN-Gremlin-1 (lanes 2) and His-tagged fl-Gremlin-1. All samples were analysed under reducing and non-reducing conditions, as indicated. MW, molecular mass (indicated in kDa). (B) Gremlin-1 interaction with BMP-2. Equilibrium state binding from BLI for ΔN-Gremlin-1 (purple squares) and fl-Gremlin-1 (pink squares) with BMP-2 data fitted to a steady-state equilibrium model shown as unbroken lines. (C) Gremlin-1 inhibition of BMP-2 in C2C12 cells. BMP-2-induced AP activity in C2C12 cells treated with BMP-2–ΔN-Gremlin-1 (purple squares) and BMP-2–Gremlin-1 (pink squares), with the fit to the IC₅₀ model shown as unbroken lines. AU, arbitrary units. E=×10 to the power indicated.

Figure 2. Crystal structure of ΔN-Gremlin-1

(A) Cartoon of ΔN-Gremlin-1 dimer with different chains coloured darker and lighter purple, and labelled to indicate parts and motifs discussed in the text. F1 and F2 indicate the fingers, W marks the wrist region and N- and C-termini are labelled N and C, respectively. (B) Close-up view of the β-sheet at the dimerization interface. Only the main chain is shown and hydrogen bonds are represented as black dashed lines. (C) Dimer of ΔN-Gremlin-1, with one of the two protomers shown with its molecular surface on to which the interaction surface between the protomers is coloured (red for oxygen atoms, blue for nitrogen atoms and yellow for carbons). (D) Close-up view of the interfacial α-helix (in transparent outline) with side chains of hydrophobic residues interacting with the helix from both protomers shown as sticks. (E) Detailed view of C141 and F143 residues at the dimerization interface.

Figure 3. Analysis of oligomeric state of ΔN-Gremlin-1 in solution

(A) MALS analysis of ΔN-Gremlin-1 dimer. Light scattering trace is shown as an unbroken line, whereas molecular mass distribution across the peak is shown by red dots. (B and C) ΔN-Gremlin-1 crystal structure shown as a ribbon and coloured surface with (B) ΔN-Gremlin-1 envelope and (C) fl-Gremlin-1 envelope shown in white.

Figure 4. BLI analysis of repetitive ΔN-Gremlin-1 interaction with BMP-2

(A) Overview of the design of the repetitive binding experiment, with all of the components shown schematically. The curves show BLI response for each of the eight channels which differed in the concentration of BMP-2 in the well, as labelled at the end point of each curve. (B) Control experiment to determine non-specific ΔN-Gremlin-1 binding to His-tagged ΔN-Gremlin-1 or the tip surface.

Figure 5. Model of ΔN-Gremlin-1 and BMP-2 oligomeric complex

(A) Complex model of 2:1 stoichiometry (two Gremlin-1 dimers: one BMP-2 dimer). (B) Complex model of 2:2 stoichiometry. (C) Model of complex oligomerization in ‘fibril-like’ manner. (D) Elongated model of ‘fibril-like’ complex formation based on crystal structure analysis. All models created ‘manually’ by orienting molecules to approximate locations and hence used for illustrative purposes only and not to be seen as precise atomic models.