Abstract
Bone morphogenetic proteins (BMPs) are antagonized through the action of numerous extracellular protein antagonists, including members from the differential screening-selected gene aberrative in neuroblastoma (DAN) family. In vivo, misregulation of the balance between BMP signaling and DAN inhibition can lead to numerous disease states, including cancer, kidney nephropathy, and pulmonary arterial hypertension. Despite this importance, very little information is available describing how DAN family proteins effectively inhibit BMP ligands. Furthermore, our understanding for how differences in individual DAN family members arise, including affinity and specificity, remains underdeveloped. Here, we present the structure of the founding member of the DAN family, neuroblastoma suppressor of tumorigenicity 1 (NBL1). Comparing NBL1 to the structure of protein related to Dan and Cerberus (PRDC), a more potent BMP antagonist within the DAN family, a number of differences were identified. Through a mutagenesis-based approach, we were able to correlate the BMP binding epitope in NBL1 with that in PRDC, where introduction of specific PRDC amino acids in NBL1 (A58F and S67Y) correlated with a gain-of-function inhibition toward BMP2 and BMP7, but not GDF5. Although NBL1(S67Y) was able to antagonize BMP7 as effectively as PRDC, NBL1(S67Y) was still 32-fold weaker than PRDC against BMP2. Taken together, this data suggests that alterations in the BMP binding epitope can partially account for differences in the potency of BMP inhibition within the DAN family.
Highlights
neuroblastoma suppressor of tumorigenicity 1 (NBL1) is a moderate antagonist important for modulating bone morphogenetic protein (BMP) signaling in vivo
Based upon our recent structure/function studies on protein related to Dan and Cerberus (PRDC), where we identified a significant portion of the BMP binding epitope of the protein, we sought to better understand how differences in affinity and specificity arise for BMP ligands across the differential screening-selected gene aberrative in neuroblastoma (DAN) family by continuing our structure/function studies on NBL1 [16]
We tested the two proteins for the ability to inhibit BMP2 signaling via the luciferase reporter assay. These results clearly show that NBL1 construct (NBL1⌬C) maintains most, if not all or more, of its antagonist phenotype when compared with the wild-type protein, where the C terminus appears dispensable for BMP2 inhibition (Fig. 2, C and E)
Summary
NBL1 is a moderate antagonist important for modulating bone morphogenetic protein (BMP) signaling in vivo. Our understanding of DAN family-mediated antagonism remains underdeveloped, despite being the largest known family of BMP antagonists in vertebrates [12] Based upon their characterized importance in numerous disease states, where different DAN proteins have been directly linked to the diseases mentioned above, there is a strong need to evaluate these proteins structurally and functionally to aid in future therapeutic design processes to restore the proper balance of BMP signaling [12]. To help fill these gaps in our understanding, we sought to determine the specificity of NBL1 trying to undermine what features in NBL1 make it a unique and mild BMP antagonist in comparison to both stronger and weaker DAN family members, including PRDC (strong) and SOST (weak) Toward this goal, we present the crystal structure of NBL1. We hope to aid in the mechanistic understanding of DAN-mediated BMP regulation
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