Zinc-Dependent Structural Stability of Human Sonic Hedgehog

Abstract

The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of </=100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.

Functional Characterization of Sonic Hedgehog Mutations Associated with Holoprosencephaly

“…This arrangement is similar to that found in several Zn 2ϩ hydrolases and has led to the hypothesis that SHH might display hydrolase activity (48,49). Denaturation studies conducted on the H140A and D147A SHH-N mutants have previously shown that the loss of Zn 2ϩ binding is accompanied by a misfolding of the proteins, which were, thus, highly susceptible to proteolytic degradation and very difficult to purify (49). One can speculate that the substitution of a histidine by a proline residue (H140P) would also impair Zn 2ϩ binding.…”

“…The crystal structure of murine Shh-N has revealed the presence of a Zn 2ϩ ion coordinated by His-141, Asp-148, and His-183 that, respectively, correspond to His-140, Asp-147, and His-182 in human SHH-N. This arrangement is similar to that found in several Zn 2ϩ hydrolases and has led to the hypothesis that SHH might display hydrolase activity (48,49). Denaturation studies conducted on the H140A and D147A SHH-N mutants have previously shown that the loss of Zn 2ϩ binding is accompanied by a misfolding of the proteins, which were, thus, highly susceptible to proteolytic degradation and very difficult to purify (49).…”

Sonic Hedgehog in Nasal Mucus is a Biomarker for Smell Loss in Patients with Hyposmia

“…It is structurally homologous to several zinc-dependent hydrolases [40] and it contains one zinc atom coordinated by two histidines and a glutamate residue [41] similar to the presence of zinc in the structure of the taste bud growth factor gustin (carbonic anhydrase (CA) VI) [42]. Elimination of zinc from either Shh or CA VI inhibits their respective activity [41,42]. CA VI and zinc components have also been shown to play a role in adenylyl cyclase metabolism by acting as a potent activator of phosphodiesterase [43].…”

Functional Analysis of Novel Sonic Hedgehog Gene Mutations Identified in Basal Cell Carcinomas from Xeroderma Pigmentosum Patients

“…Day et al (36) analyzing the zinc coordinating residues of SHHN have shown that D147 and H140 are important for maintaining full zinc occupancy. Modifications of these residues to alanine, D147A and H140A, resulted in a substantial reduction in protein stability, which was undetectable at the end of the tests when assayed for C3H10T1/2 differentiation or in the neural plate explant assay, suggesting proteolytic degradation (36). The mutated SHH protein we have detected in a BCC modifying the same residue, D147N, was also found to be unstable when expressed in bacterial or mammalian cells.…”

“…It is still unclear whether this protein domain is implicated in the postulated hydrolase activity or if it is required for stabilizing the folded protein structure (36). Day et al (36) analyzing the zinc coordinating residues of SHHN have shown that D147 and H140 are important for maintaining full zinc occupancy.…”