Zinc-dependent structural stability of human Sonic hedgehog

The role of the zinc site in the N-terminal fragment of human Sonic hedgeho g (ShhN) was explored by comparing the biophysical and functional propertie s of wild-type ShhN with those of mutants in which the zinc-coordinating re sidues 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 E176 A mutant proteins retained 1 mol of zinc/mol of protein after extensive dia lysis, 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 pro teins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were sig nificantly less active. These assays also indicated that the H140A and D147 A mutants were susceptible to proteolysis. CD, fluorescence, and H-1 NMR sp ectra 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 measur ements at 37 degrees C showed evidence of some structural differences in th e H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation stu dies revealed that the loss of zinc from the H140A and D147A mutants destab ilized the folded proteins by similar to 3.5 kcal/mol, comparable to the ef fect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal m elting curves of wild-type ShhN gave a single unfolding transition with a m idpoint T-m of similar to 59 degrees C, whereas both the H140A and D147A mu tants 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. Addit ion of zinc to the H140A and D147A mutants resulted in a partial restoratio n of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assa y that indicated that they bound zinc with K-d values of similar to 1.6 and similar to 15 nM, respectively, as compared to a value of less than or equ al to 100 pM for wild-type ShhN. The properties of the E176A mutant were in distinguishable from those of wild-type ShhN in all biophysical and functio nal assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hy drolase activity for in vitro biological function.