Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

Abstract

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

“…The ability of Shh-E177A to induce the Hh response when expressed ectopically in the developing brain could be explained by cell-autonomous signaling. Purified ShhN-E177A is reportedly more stable in solution than ShhN, supporting the notion that Shh has an intrinsic cannibalistic peptidase activity (Rebollido-Rios et al, 2014). Perhaps unsurprisingly, the zinc coordination domain is found mutated in some individuals with the Shh signaling-related birth defect holoprosencephaly (Roessler et al, 1996;Traiffort et al, 2004), further indicating that the putative peptidase activity of Shh is important for normal function.…”

“…The overall structure of ShhN and the BacHhs indicate that they consist of a regulatory calcium-binding and a catalytic zinc coordinating domain (Rebollido-Rios et al, 2014), making up most of ShhN outside the extreme N-terminal Ptch1-binding domain. With the exception of BacHhs, bacterial M15A metallopeptidases lack the Hh/BacHh-type calcium coordination domain, and this domain is thus unlikely to be required for catalytic function per se.…”

“…showing that the N-terminal 22 residues of Shh that are not part of the peptidase domain, mediate binding to Ptch1 (Gong et al, 2018;Qi et al, 2018a;2018b) and suffice to regulate Ptch1 activity (Tukachinsky et al, 2016). Some bacteria have genes coding for peptidases that coordinate zinc and calcium identically to Shh (Rebollido-Rios et al, 2014;Roelink, 2018). These bacterial peptidases (members of the M15A subfamily of zinc D-Ala-D-Ala carboxypeptidases) cleave murein peptidoglycans, suggesting that Shh too might cleave a glycan-modified protein, possibly a matrix heparan sulfate proteoglycan (HSPGs).…”

“…Possible mechanisms of catalysis of zinc peptidases have been elucidated with the help of structural models of enzyme-inhibitor complexes. Thermolysin is a well-studied zinc metallopeptidase structurally related to Shh (Hall et al, 1995;Rebollido-Rios et al, 2014). Shh and Thermolysin coordinate zinc via two histidine and an aspartic acid residue.…”

Gain-of-function Shh mutants activate Smoin cisindependent of Ptch1/2 function

“…Shh-E90 and -H183 are involved in the binding of Ca 2+ and Zn 2+ metal ions, respectively, and the coordination of the Zn 2+ ion by H141, D148, and H183 (the "triad", Figure 6A) resembles that of Zn 2+ peptidases, (Hall et al, 1995a) including Thermolysin (Rebollido-Rios et al, 2014). Hhs belong to a larger family of DD-transpeptidases (van Heijenoort, 2011) that has many members in prokaryotes.…”