Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

Abstract

The Hedgehog pathway is an essential cell-signaling paradigm implicated in cancer tumorigenesis and the developmental disorder holoprosencephaly, making it an attractive target for therapeutic design. The N-terminal domain of the Sonic Hedgehog protein (Shh-N) is the essential signaling molecule in the Hedgehog pathway. In this role Shh-N interacts with its cognate membrane receptor Patched, as well as the regulatory proteins HHIP and CDO, by utilizing interfaces harboring one or more divalent ions. Here, the crystal structure of human Shh-N is presented at 1.43 Å resolution, representing a landmark in the characterization of this protein. The structure reveals that the conserved Zn 2+binding site adopts an atypical octahedral coordination geometry, whereas an adjacent binding site, normally occupied by binuclear Ca 2+ , has been supplanted by a single octahedrally bound Mg 2+ . Both divalent sites are compared with those in previous Shh-N structures, which demonstrates a significant degree of plasticity of the Shh-N protein in terms of divalent ion binding. The presence of a high Mg 2+ concentration in the crystallization medium appears to have influenced metal loading at both metal ion-binding sites. These observations have technical and design implications for efforts focused on the development of inhibitors that target Shh-N-mediated protein-protein interactions.

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

“…Both structures show similar ion-to-oxygen coordination distances of 2.46 ± 0.10 for dmU1A and 2.50 ± 0.05 Å for dmU1A(F37M/F77M). Given the identical number of electrons for Mg 2+ and Na + , crystallization from 2.2 M Na-malonate, and the observation of slightly longer ion-to-oxygen coordination distances for Na + (2.53 ± 0.17 Å) [103] compared to Mg 2+ (2.06 ± 0.02 Å) [104,105], it appears that R70 is a nexus for Na + coordination in the dmU1A structure. It has been noted that Na + is frequently mistaken for Mg 2+ in crystal structures, and the greater formal charge of alkaline earth metals is frequently associated with coordination by one or more acidic ligands [100].…”

Hedgehog signaling in tissue homeostasis, cancers, and targeted therapies

“…Dhh and Ihh also undergo autocatalytic cleavage to form Dhh-N and Ihh-N, in which the C-terminal structural domain is removed. 46 This N-terminal structural domain is then modified by N-terminal palmitoylation as well as the cholesterol addition of C-terminal amino acids, resulting in the formation of the mature proteins Dhh-N and Ihh-N. 47 All three HH ligands share high sequence homology, similar overall fold and are capable of binding to and activating the same signaling pathway receptors. They mainly differ in their N-terminal amino acid sequence; however, their N-terminal sequence similarity may also reach 76–91%.…”