Journal: The FASEB Journal

Article Title: Prodomain processing controls BMP ‐10 bioactivity and targeting to fibrillin‐1 in latent conformation

doi: 10.1096/fj.202401694R

Figure Lengend Snippet: SPR affinity data for interactions between immobilized BMP‐9, ‐10, and ‐9/10 fusion PDs and flowed over BMP‐10 GF.

Article Snippet: For western blot and sandwich ELISAs the following antibodies were used: anti‐His 6 ‐HRP (#130‐092‐785, Miltenyi Biotec, Germany), goat anti‐human BMP‐10 propeptide (#AF3956‐SP, R&D Systems, Minneapolis, MN, USA), mouse anti‐human BMP‐10 GF (#MAB2926, R&D Systems), rabbit anti‐human phospho‐SMAD 1/5/9 (pSMAD1/5/9) (#13820, Cell Signaling Technology, Danvers, MA, USA), mouse anti‐rabbit GAPDH (#ab8245, Abcam, Waltham, MA, USA), and rabbit anti‐human BMP‐7 GF (#500‐P198, PeproTech, Rocky Hill, NJ).

Techniques:

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a One asymmetric unit of the BMP10:BMPRII 1.48 Å crystal structure. Chain identities (IDs) A to D are labelled. BMP10 is coloured in coral and light purple, BMPRII coloured in green. b 2.4 Å structure of BMP10:BMPRII with chain IDs labelled. Only one monomer of BMP10 (in coral) and BMPRII (in green) in an asymmetric unit. One symmetry-related molecule is shown in grey to illustrate the BMP10 dimer bound to two copies of BMPRII. c – e Overall structure of the ALK1:BMP10:BMPRII complex. Four copies of each BMP10, ALK1 and BMPRII monomers are found in one asymmetric unit, forming two copies of ternary signalling complexes shown in semi-transparent yellow and grey surface. Chain IDs in complex 1 (cpx1) ( d ) and cpx2 ( e ) are shown. In cpx1, BMP10 monomers are coloured in coral and light purple, ALK1 in yellow and BMPRII coloured in green. In cpx2, BMP10 monomers are coloured in coral and cyan, ALK1 in dark yellow, BMPRII in green. f An illustration of BMPRII-signalling complex in relation to cell surface. The last residues in ALK1 and BMPRII ECD cDNA-encoded sequences are 118 and 150, respectively. The last residues that can be seen in the crystal structures are shown in spheres and labelled. The 1.48 Å BMP10:BMPRII structure (in grey and semi-transparent) is superimposed on the ternary signalling complex (coloured as in Fig. 1d, cpx1) to show positions of further modelled sequence in BMPRII C-termini. The C-terminal 10–13 residues in both ALK1 and BMPRII that are not visible in the structure are represented by thick dashed lines.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Sequencing

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a Overlay of BMP10:ALK1 from the ternary signalling complex (cpx1, magenta, cpx2, purple) to those from binary complexes (PDB code 6SF1 in grey; 6SF3 in cyan for BMP10 and orange for ALK1). The backbones of all overlaid molecules are shown in ribbon. BMP10 from 6SF3 also shown in semi-transparent cartoon. Because in both 6SF1 and 6SF3, there was only one copy of BMP10:ALK1 monomer in an asymmetric unit, the dimeric receptor complexes for 6SF1 and 6SF3 were generated with a symmetry-related molecule and the two BMP10:ALK1 interfaces in 6SF1 and 6SF3 dimer would be identical. b Comparison of the buried surface area at the BMP10 and ALK1 interface in binary and ternary receptor complexes. c Overlay of all ALK1 chains, displayed in ribbon on BMP10 surface (light cyan). Four parts of BMP10 binding sites on ALK1 identified previously are highlighted by dashed lines. The colour for each chain is shown below. d Zoomed-in views of ALK1 H87 and E59 interaction area. H-bond interactions are shown with dotted lines. Same colour scheme as in c . The only interactions can be seen are in grey (from 6SF1), and orange and cyan (from 6SF3). Detailed information and the list of the interactions can be found in Supplementary Table .

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Generated, Comparison, Binding Assay

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a,b Secondary structural elements of the type II receptor ( a , represented by BMPRII) or BMP ( b , represented by BMP10), coloured in rainbow from blue at the N-terminus to red at the C-terminus. c , d Overlay of seven BMP10:BMPRII 1:1 complexes by BMPRII ( c ) and a zoomed-in view of the wrist helix region ( d ). e Overlay of seven BMP10:BMPRII 1:1 complexes by BMP10 wrist helix. In c – e , complex AI in green, CK in magenta, BJ in cyan, DL in dark yellow, AC in light purple, AB in dark blue, BD in grey. In c , e , red arrows highlight the movement of BMP10 among the 7 complexes, whilst the green arrow highlights the movement of BMPRII among the 7 complexes. f , g Overlay of complexes CK (magenta) and BD (grey) by BMP10 wrist helix. f . The hinge region and the distances between the BMPRII finger 2 hairpins and BMP10 fingertips 3/4 are shown. g A zoomed-in view of the hinge area, showing the rotation angle between complex CK V406/CA, complex BD K410/CA and complex BD V406/CA is 22.7 degrees.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques:

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a BMP10:BMPRII binding interface in complex AC. BMP10 (in coral)-binding surface on BMPRII (purple) can be broadly divided into three regions. The central hydrophobic triad (Y67, W85 and F115), the β4 strand to the A-loop (green oval), the F3-loop and the region connecting the A-loop and the F3-loop (light blue circle and orange oval). b – f Detailed interactions between BMP10 and BMPRII, at the β4 strand and A-loop region in all complexes ( b ), at the F3-loop ( c ) and the regions connecting the A-loop and the F3-loop in complex AC ( d ), at BMPRII S107 region in complex BD ( e ) and AB ( f ). BMP10 is shown in coral sticks throughout b – f , whereas BMPRII is in grey for complex BD, in dark blue for complex AB, in yellow for complex DL, in magenta for complex CK, in cyan for complex BJ and in green for complex AI. In a – f red dashed lines denote H-bonds, with distance all between 2.7-3.7 Å if not labelled. Underlined residue numbers are those from BMP10, and residues numbers in normal text are those from BMPRII. g Buried interface area between BMP10 and BMPRII in binary and ternary receptor complexes. *Complex BD has much smaller buried surface area because some sidechains at the interface were deleted between the A-loop and F3-loop due to poor densities.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Binding Assay, Residue

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a Sequence alignment of BMPRII ECD and ActRIIB ECD. Residues at the binding interface with BMP10 or BMP9 are shown in blue. Residues that are not modelled in the BMPRII structure are shown in grey. Hydrophobic triad residues are highlighted in cyan. Positions of the four loops are highlighted below the sequence. Lines above the sequence highlight the residues deleted in the mutagenesis studies. ΔF1(F3) = deletion of finger 1 (finger 3) residues; ΔGDP = deletion of residue Gly, Asp and Pro. b , c BMP10-binding site on BMPRII ( b , light purple) and BMP9-binding site on ActRIIB ( c , cyan). Residues making direct interactions with BMP10 or BMP9 are coloured in magenta, with hydrophobic triad residues shown in sticks. BMP10 G89 is highlighted in a magenta sphere. Water molecules that mediate hydrogen bond interactions between BMPRII and BMP10 are shown in yellow spheres. d BMPRII in binary (complex AC, purple cartoon) and ternary (complex AI, green cartoon) complexes on BMP10 surface (pale blue), showing G89 (magenta spheres) docking into a deep pocket on BMP10. e ActRIIB (cyan cartoon) on BMP9 surface (pale blue, from PDB entry 4FAO) does not have A-loop mediated interaction. In d , e , residues making H-bond interactions shown in magenta, and hydrophobic triad shown in magenta sticks.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Sequencing, Binding Assay, Mutagenesis, Residue

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a An overlay of all BMP10 monomers from different protein interaction contexts. Sources of structures are listed in Supplementary Table . b A close-up view of fingertip 3/4 with all the structures annotated. c A closed-up view of fingertip 3/4 region after superposition of all BMP9 monomers from different protein interaction contexts. Sources of the structures are listed in Supplementary Table . The overlay of the full monomers can be found in Supplementary Fig . d A schematic diagram depicting the context-dependent conformation of fingertip 3/4 in BMP9 and BMP10.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques:

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a The sensorgram of monomeric BMPRII WT ECD binding to BMP10 immobilised on a CM5 Biacore chip. Control experiments of ALK1-Fc and BMPRII-Fc binding to the same BMP10 chip are shown in Supplementary Fig . b G89-containing A-loop is essential for BMPRII binding to BMP10. c F1-loop and F3-loop deletion mutants binding to BMP10. d Locations of PAH mutations on the BMPRII ECD structure (purple cartoon). Red spheres: residues predicted to be deleterious; cyan spheres, residues predicted to be benign. e Sensorgrams of BMPRII ECD proteins containing PAH mutations binding to BMP10. f . Summary of kinetic parameters from the Biacore binding experiments. *G89A has also been found in an hereditary PAH case, but predicted to be a benign mutation. WT wild type, ECD extracellular domain, ΔF1(F3) deletion of finger 1 (finger 3) residues, ΔGDP deletion of Gly, Asp and Pro in the A-loop. M Molar concentration, s = second, k a = association rate constant, k d = dissociation rate constant, K D = k d /k a .

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Binding Assay, Control, Mutagenesis, Concentration Assay

Journal: Nature Communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: a A representative native PAGE of prodomain displacement experiment. b Quantification of native PAGE results. The band intensities of Pro:BMP10 and prodomain were obtained by densitometry using ImageJ. The ratio of Pro:BMP10/prodomain for each lane was calculated, then normalised to that of the WT control run on the same gel. Each mutant protein was run on at least three independent native PAGE experiments, with two WT controls on each gel to allow normalisation. Final N numbers for each sample are labelled below the graph. Conditions with BMPRII ECDs at 2-fold excess (2x) are shown in open bars, and those with 5x in hashed bars. The two dotted lines mark the mean values for 2x and 5x WT BMPRII, respectively. Data are presented as mean values + /− SEM. One-way ANOVA analysis for WT ECD group, **** P < 0.0001, ** P < 0.01; for BMPRII at 2x excess group, Dunnett’s post test comparing with WT BMPRII ECD, $ P < 0.05, $$ P < 0.01; for BMPRII at 5x excess group, Dunnett’s post test comparing with WT BMPRII ECD, # P < 0.05, ### P < 0.001, #### P < 0.0001. c Schematic depicting the experiment of the prodomain displacement by ELISA. d Quantitative results of prodomain displacement by ELISA. All readings are normalised to the WT on the same plate. N = 5 for WT ECD, N = 3 for each mutant protein. Each N number represents an independent ELISA experiment. Data are presented as mean values + /− SEM. One-way ANOVA analysis for BMPRII at 125x excess, with Dunnett’s post test comparing with WT BMPRII ECD (mean value shown as a dotted line). # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001. In b , d , coloured in red are residues near the hydrophobic triad, in orange are residues from the A-loop, in yellow are F3-loop; in brown, non-interface residues, and in purple, F1-loop which is not visible in the structure. Source data, including the exact p values for b , d , are provided as a Source Data file. ECD = extracellular domain, GF-domain = growth factor domain, Pro:BMP10 = non-covalent complex of BMP10 prodomain with its GF-domain.

Article Snippet: After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody (0.04 μg/well, cat. No. BAF3956, R&D Systems) in 1% BSA/PBS was added.

Techniques: Clear Native PAGE, Control, Mutagenesis, Enzyme-linked Immunosorbent Assay