Use of MAD to Solve Crystal Structures of Selenomethionyl-Substituted Fragments of the Developmental Signaling Molecule Hedgehog. Daniel J Leahy, Traci M.T. Hall, Jeffery A. Porter, Keith E. Young, and Philip A. Beachy.

Within the past few years, members of the Hedgehog (HH) family of secreted signaling molecules have emerged as the primary signals generated by certain embryonic patterning centers. Hedgehog proteins undergo an autoprocessing in which the full-length 45 kd protein is broken down into a 19 kd N-terminal domain that possesses all known HH signaling activity and a 26 kd C-terminal domain responsible for the autoprocessing. The processing is not limited to a simple cleavage reaction, but also results in the covalent attachment of a cholesterol moiety to the N-terminal signaling domain. This attached cholesterol tethers the signaling domain to the plasma membrane and is essential for appropriate HH function. HH proteins represent the first example of a protein covalently modified with cholesterol, but several proteins with domains homologous to the HH C-terminal domain that have unrelated N-terminal domains have been identified suggesting that other proteins may be similarly modified. We have solved crystal structures of selenomethionyl-substituted forms of both the N- and C-terminal domains of a hedgehog protein by MAD. Experiences with the MAD technique as well as implications of the structures for HH signaling and biogenesis will be discussed.