Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

Abstract

Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10 -20-fold to tighter than 1 nM primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn 2؉ cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.

Dual Roles of the Cardin-Weintraub Motif in Multimeric Sonic Hedgehog

“…In contrast, site-directed mutagenesis of CW residues that mediate HS binding of the monomeric morphogen, such as Arg 34 and Lys 38 (16), did not abolish HS binding and biological activity of the physiologically relevant, multimeric morphogen. Analysis of the human Shh crystal structure (Protein Data Bank entry 3M1N (23)) provided an explanation for this finding.…”

“…B and C, detailed view of the interaction site. CW residues Lys 33 , Arg 35 , and Lys 39 (mouse nomenclature) are surface exposed, whereas residues Arg 34 and Lys 38 make contact with the negatively charged patch on the adjacent molecule. D, same orientation as C, with the interacting molecule removed for better visualization of residues Arg 34 and Lys 38 .…”

“…CW residues Lys 33 , Arg 35 , and Lys 39 (mouse nomenclature) are surface exposed, whereas residues Arg 34 and Lys 38 make contact with the negatively charged patch on the adjacent molecule. D, same orientation as C, with the interacting molecule removed for better visualization of residues Arg 34 and Lys 38 . that CW residues Lys 33 , Arg 35 , and Lys 39 modulate ShhNp biological activity in two cell-based bioassays for Shh activity; however, they are not required for ShhNp activity in these experimental settings.…”

“…What is the role of CW residues Arg 34 and Lys 38 ? In this work, we demonstrate that site-directed mutagenesis of these CW residues abolished HS binding of the monomeric morphogen but affected HS binding of the mutant multimer to a lesser degree.…”

Two Distinct Sites in Sonic Hedgehog Combine for Heparan Sulfate Interactions and Cell Signaling Functions

“…Purified hShhNC24II proteins were added to the culture medium the day after plating at concentrations of 300, 500, 1000, or 2000 ng/ml. Monoclonal antibody 5E1 is a specific inhibitor of the Shh pathway by binding to Shh and neutralizing its activity (30). To some samples, 5E1 (10 g/ml) was added together with hShh proteins.…”