Schematic domain organization of human endoglin and ALK1 and western blots of endoglin domains.

<p>Bar diagram of (A) endoglin and (B) ALK1 with the domains indicated and highlighted in different styles. TM, transmembrane region, ZP, zona pellucida. The putative Asn glycosylation sites Asn88, Asn102, Asn121, Asn134 and Asn306 of endoglin and Asn98 of ALK1 are labelled within green ovals. The constructs used in this study and the domains encompassed by these are shown below the bar diagram of the respective full length protein. (C) Western blots of endoglin constructs. Endo₃₃₈, Endo₃₆₂ and LG-Endo_EC were analyzed by 10% SDS polyacrylamide electrophoresis gel followed by western blotting with an anti-His₆ antibody. Samples reduced with dithithreitol (DTT) were incubated for 1 h with 10 mM of this reagent at 65°C. All samples (0.5 µg of protein) were then denaturated by boiling at 95°C for 5 minutes prior to charging onto the gel. The molecular weight markers (M) are indicated at the left of the samples. For both Endo₃₆₂ and LG-Endo_EC, dimeric species are visible, while Endo₃₃₈ was only observed in monomeric form.</p

Experimental and modelling SAXS parameters.

a<p>Values for I(0) have been extrapolated by the Guinier approximation from the experimental scattering profiles.</p>b<p>Concentration of the protein used for the calculation of the estimated Mw.</p>c<p>Relative molecular mass estimated from I(0) and the concentration of the protein through BSA calibration.</p>d<p>The Porod volume was calculated using PRIMUS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029948#pone.0029948-Mertens1" target="_blank">[41]</a>.</p>e<p>Expected molecular mass predicted from the sequence and assuming full occupation of the glycosylation sites.</p>f<p>Rg (Guinier), Rg (GNOM), radius of gyration given by the Guinier approximation, and calculated by the program GNOM, respectively, given in nm.</p>g<p>Maximum dimension of the macromolecules. χ²^(over) Discrepancy between the SAXS profile and its fit by the overall shapes-models calculated by DAMMIF, and χ^{2 (sasref)} the average discrepancy of the best atomic models estimated with the program CRYSOL. ND, not determined.</p

Functional analysis of recombinant endoglin and ALK1 proteins.

<p>ID1 expression in BMP-9 stimulated HMEC-1 cells after 36 hours in the presence or absence of LG-ALK1_EC, Endoglin₃₃₈ or LG-Endo_EC. The ALK1 and endoglin ectodomains hijack BMP-9 and therefore signaling is diminished. Endo₃₃₈ only partially inhibit signaling, due to a less stable complex formed between the orphan domain and the cytokine. (*) Statistically significant (p<0.01) difference compared to unstimulated control cells (Ctl). (#) Statistically significant (p<0.01) difference compared to ID1 expression of BMP-9 stimulated cells.</p

Functional interactions between endoglin, ALK1 and BMP-9.

<p>The interactions of (A) Endo_EC, (B) ALK1_EC, (E) Endo₃₃₈ and (F) Endo₃₆₂ with BMP-9 were investigated by SPR. While HG-Endo_EC and Endo₃₆₂ dissociated slowly, Endo₃₃₈ dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-Endo_EC and HG-ALK1_EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-Endo_EC (C, left) or HG-ALK1_EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2^nd ligand. Both HG-ALK1_EC (C, right) and HG-Endo_EC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (k_a) and dissociation (k_d) rate constants and the dissociation affinity constant (K_D).</p

Analysis of ligand binding to BMP-9 assessed by Surface Plamson Resonance.

<p>Kinetic analysis of endoglin and ALK1 binding to BMP-9 was performed in triplicates on a Biacore T100 at 25°C as described in the experimental procedures. Data were globally fit to a 1∶1 binding model using the Biacore T100 evaluation software.</p