Identification and structural characterization of heparin-binding hotspots in Gremlin
Melissa Buelt-Gebhardt,1 Laurie L. Shekels,1,2 James Curry,1 Matthew S. Nelson,1 Mark A.
Klein,1,2
3
Yuk Y. Sham, and Pankaj Gupta1,2
1
Hematology/Oncology Section, Minneapolis VA Health Care System, Minneapolis, MN 55417.
Hematology/Oncology/Transplantation Division, Department of Medicine, and 3Center for Drug
Design; Academic Health Center, University of Minnesota, Minneapolis, MN 55455
2
Abstract
Background: Gremlin1 is a multi-functional, heparin/heparan sulfate (HS) binding protein
belonging to the cystine knot superfamily of bone morphogenetic protein (BMP) antagonists that
plays a pathogenetic role in a remarkable variety of malignancies as well as diseases of the
lungs, kidneys, eyes and bones. However, there are no drugs that target this molecule. Binding
interactions with HS are essential for critical pathophysiological effects of Gremlin1. Similarly, a
common feature of many proteins, receptors and pathways influenced by Gremlin1 is that their
functional activity is modulated by interactions with HS. A prototypical example is that
competitive inhibition of binding of Gremlin1 to cell surface HS prevents its binding to vascular
endothelial growth factor receptor (VEGFR)-2, and decreases angiogenesis. Inhibiting
Gremlin1-HS interactions may therefore be of broad therapeutic value.
Methods: To elucidate the structural features of its heparin-binding domains, we performed sitedirected mutagenesis and homology modeling of Gremlin1, together with molecular dynamics
simulation of the Gremlin1-heparin complex. Changes in heparin-binding affinity of mutated
Gremlin1 were measured using surface plasmon resonance. Relative cell surface binding ability
of the mutated Gremlin1 proteins was evaluated using flow cytometry.
Results: Site directed mutagenesis of clusters of basic amino acids followed by surface plasmon
resonance binding studies identified a previously unrecognized heparin-binding site (“region A”)
upstream of the cystine knot and BMP binding domain. Homology modeling indicated that this
site is located on the surface of an alpha helical region of Gremlin1 near the cystine knot located
in the central core. Molecular dynamics simulation analysis identified a second previously
unrecognized group of basic amino acids located C-terminal of the BMP binding domain that are
predicted to interact with heparin. Of note, both groups of basic amino acids are involved in
each of the two possible complexes that can form between Gremlin1 and heparin. Cell surface
binding studies confirmed that replacement of basic amino acids by alanine in region A
markedly reduces the ability of Gremlin1 to bind to the surface of malignant cells. Studies
examining the relative functional effects of the mutated Gremlin1 proteins are underway.
Conclusions: We identified a previously unrecognized group of basic amino acids in Gremlin1
that constitutes a major heparin/HS-binding “hotspot.” Our characterization of heparin/HSbinding domains of Gremlin1 may provide a basis for structure-based design of novel
pharmacologics that target this pathogenic protein.