Hedgehog pathway antagonist 5E1 binds hedgehog at the pseudo-active site

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Proper Hedgehog (Hh) signaling is vital for embryonic development and tissue repair.
• Aberrant Hh pathway activity is linked to various cancers.
• The 5E1 antibody is a known inhibitor of Hh pathway ligand binding to Patched1 (Ptc1).

Purpose of the Study:

• To engineer and characterize a chimeric 5E1 (ch5E1) antibody for Hh pathway inhibition.
• To investigate the binding properties of ch5E1 to Sonic hedgehog (Shh).
• To elucidate the structural basis of ch5E1-Shh interaction.

Main Methods:

• Protein engineering of a chimeric 5E1 antibody.
• Biochemical and biophysical assays to assess binding affinity and kinetics.
• X-ray crystallography to determine the co-crystal structure of Shh bound to ch5E1 Fab fragment.

Main Results:

• Engineered ch5E1 retains Hh-binding properties similar to the original 5E1 antibody.
• Calcium ions enhance ch5E1 binding to Shh, akin to regulatory receptors Cdon and Hhip.
• ch5E1 exhibits a 10-20 fold increase in binding affinity for Shh in the presence of calcium and zinc ions, primarily due to a reduced dissociation rate.
• The co-crystal structure reveals ch5E1 binds Shh at the pseudo-active site, overlapping with the Hhip binding site.
• Unlike Hhip, ch5E1 does not directly coordinate zinc ions in the pseudo-active site.

Conclusions:

• The engineered ch5E1 antibody is a potent inhibitor of Shh.
• Metal ions, particularly calcium, play a significant role in modulating ch5E1-Shh interactions.
• The structural data provides a molecular understanding of antibody-mediated inhibition of Shh, distinct from natural antagonists.
• This study presents the first structure of an inhibitory antibody bound to a metalloprotease fold in the Hh pathway.