Structural and functional insights into endoglin ligand recognition and binding

Abstract

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Figure 1. Schematic domain organization of human endoglin and ALK1 and western blots of endoglin domains.

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.

Figure 2. Functional interactions between endoglin, ALK1 and BMP-9.

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).

Figure 3. Functional analysis of recombinant endoglin and ALK1 proteins.

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.

Figure 4. SAXS data analysis and rigid body modeling.

(A) The experimental SAXS profile of recombinant Endo₃₃₈, measured at the ID14-3 BioSAXS beamline. The Kratky plot (inserted), displays a gaussian behavior, which indicates the well folded nature of the construct. (B) The distance distribution function, p(r) obtained from GNOM with a D_max of 9.4 nm also suggests a compact structure. (C) The orphan domain model obtained from I-TASSER together with 5 Man₅NAG₂ glycans, restricted by their attachment sites was fitted by rigid body modeling with SASREF, yielding a fit of the model (red line) against the experimental Endo₃₃₈ scattering profile (blue crosses) of χ² = 1.1. The input models used, are depicted above the fit curve. (D) The resulting model is displayed as a cartoon in three orthogonal views, with the attached sugars depicted as balls, fitted into the molecular envelope from DAMMIF.