Structure of the Alk1 Extracellular Domain and Characterization of Its Bone Morphogenetic Protein (BMP) Binding Properties

Abstract

Bone morphogenetic proteins (BMPs) are secreted signaling proteins – they transduce their signals by by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homolog Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the β-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the β4-β5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the β4-β5 loop, and to lesser extent β1-β2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9:Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3, but is rotated by 40 degrees. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the β4-β5 loop that contribute significantly to binding and are non-conservatively substituted relative to the corresponding residues in Alk3.

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily

“…The structurally characterized receptor ectodomains of the TGF-β superfamily, which includes the type I receptors Alk1 [55,56], Alk3 [11,57], Alk5 [10,12,58], and Alk6 [59] and the type II receptors ActRII [8,60], ActRIIb [13,61], BMPRII [62], and TβRII [63–65], have each been shown to adopt a three finger toxin fold, consisting of three pairs of antiparallel β-strands stabilized by a characteristic pattern of four disulfides (Fig. 1C).…”

“…The majority of receptors have been produced as secreted proteins, either alone as monomers, or as dimers by fusing them with the Fc region of an antibody. The Fc-dimerized receptors typically bind their cognate signaling proteins with affinities that are 1 – 2 orders of magnitude greater than their non Fc-dimerized counterparts [55,66,74], which is expected for a bivalent interaction between a covalently dimerized receptor and a covalently dimerized signaling protein. The Fc-dimerized receptor ectodomains are useful for increasing potency for use in binding studies [66] or as inhibitors in cells or animals [75–77].…”

“…The ectodomains, if expressed alone, form highly refractile inclusion bodies and must be renatured [10,52,55,58,64,73,79]. The overall procedure parallels that used for the signaling proteins, including initial isolation of the inclusion bodies, subsequent purification of the reduced protein in denaturant, and folding by dilution into non-denaturing buffers that include redox-active species that promote disulfide exchange.…”

Activin receptor-like kinase 1 as a target for anti-angiogenesis therapy

“…Recently the BMP-9 binding domain of ALK1 was analysed. ALK1 and endoglin non-competitively bind BMP-9, indicating that endoglin and ALK1 together capture BMP-9 on the cell surface by using different sites for BMP-9 interaction [67]. ALK1 interacts with TGF-b1 and TGF-b3 in the presence of TbRII and with BMP-9 in the presence of activin receptor (ActR)IIa or BMPRII [67].…”

ALK1 signaling in development and disease: new paradigms

“…More recently, BMP9 and BMP10 have emerged as physiologically relevant ALK1 ligands (Figure 1). BMP9 and BMP10 growth factor domain homodimers are the only TGFβ family ligands that bind ALK1 with high affinity [101, 111–113]. BMP9 is synthesized primarily by the liver, with transcripts reported in hepatocytes, biliary cells, ECs, Kupffer cells, and stellate cells [114, 115].…”