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.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Recombinant chimeric 5E1 (ch5E1) was expressed in Chinese hamster ovary cells and purified by protein A and gel filtration chromatography. ch5E1 was comparable with the parental m5E1 antibody in recognizing cell surface Sonic hedgehog (Shh) stably expressed in COS cells and endogenous Hh produced by HT29 cells as analyzed by flow cytometry (Fig. 1, B and C)
Both antibodies bind to the same site on Shh, because they compete with each other for Shh binding on the cell surface (Fig. 1D). ch5E1 retained its specificity for Hh, staining the notochord, floor plate (Fig. 1E), and gut of developing mouse embryos by immunofluorescence, in agreement with previous data [40, 41]
Summary
Sonic hedgehog; Hh, hedgehog; Dhh, Desert hedgehog; Ihh, Indian hedgehog; Ptc, Patched; Hhip, Hedgehoginteracting protein; CDR, complement determining region; ITC, isothermal titration calorimetry; PBS, phosphate-buffered saline. The structures of the N-terminal signaling domain of Shh (hereafter referred to as Shh, known as Shh-N) bound to Hhip [20, 21] and Cdon [11] revealed that their respective binding sites are centered on the Shh pseudo-active site and the adjacent Ca2ϩ-binding site. We determined the x-ray crystal structure of ch5E1 Fab alone and in complex with human Shh and found that 5E1 blocks access to the pseudo-active site groove on Shh. Notably, the 5E1 epitope on Shh largely overlaps with the binding site of the natural Hh antagonist receptor Hhip, which we recently showed competes with Ptc for Shh binding [21]. These data explain the molecular basis of 5E1 inhibition of the Hh-Ptc interaction
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