The Sonic hedgehog protein (Shh) N-terminal domain (Shh-N) (PDB ID=3M1N) in humans is the most extensively characterized form that belongs to the hedgehog protein family (1, 2). The other two mammalian homologs have also been characterized and are referred to as Indian hedgehog (Ihh) and Desert hedgehog (Dhh) (2). Hedgehog proteins are extracellular signaling molecules with critical roles in embryonic development in both vertebrates and invertebrates (2, 3).

Shh regulates morphogenic processes including neural tube pattern formation and determination of central nervous system polarity (3). Null mutation of murine Shh gene results in holoproscencephaly, the lack of differentiation in the prosencephalon resulting in a common scleral rim much like that of the eponymous Sonic the Hedgehog, a popular video game character from the 1990s (3). Shh is not only a local short-range signaling molecule but also a long range morphogen (2). It is important to further characterize Shh in order to continue elucidating embryonic development and understand and accommodate the diseases associated with Shh mutation (2, 3). Recent studies have also implicated the anti-apoptotic role of Shh in certain cancer formation including adrenocortical tumors, hepatocellular carcinoma, and ameloblastoma (4).

Initially, Shh is synthesized as a 45-kDa precursor that is cleaved autoproteolytically into two secreted peptides, the 19-kDa Shh-N and 26-kDa Shh-C (2, 3, 5). According to the ExPASy isoelectric point and molecular weight calculator, the theoretical molecular weight and isoelectric point, based on amino acid sequence, of Shh-N are 19,683.16 kDa and 9.00 (pI) respectively (5).  ExPASy (Expert Protein Analysis System) is the bioinformatics resource portal currently operated by the Swiss Institute of Bioinformatics (SIB) that provides both vertical and horizontal integration for different fields of life science including proteomics, genomics, system biology, and phylogeny (5). 

It is believed that the Shh-C peptide mediates autoproteolysis, which is followed by dual lipid modification responsible for the addition of a cholesterol moiety at the C-terminal followed by the addition of a palmityl moiety at the N-terminal, of the Shh-N fragment (2, 3, 6). Null mutation blocking autoproteolysis and cholesterylation leads to impaired Shh function suggesting that the Shh-N alone is responsible for mediating signaling and dual lipid modification (3). Dual lipid modification has been implicated in long range Shh signaling (3). The cholesterol moiety is responsible for guiding the Shh-N in carrying out its morphogenic functions, specifically the determination central nervous system polarity. Shh-N functions in multimeric units that undergo palmitoylation-dependent cleavage to become functional, soluble multimers (2, 7). Autoproteolysis and dual lipid modification occur before secretion, which is a characteristic unique to Shh within its family (2).

Shh signaling is induced by binding of the Shh-N to a Patch (Ptc) mediated Smoothened (Smo) transmembrane receptor (Ptc-Smo) (3). Binding of Ptc reverses repression of Smo and allows an event of signaling cascade to occur (2). By mutagenizing certain suitable residues into cysteine residues and subsequently applying various thiol modifications, it is possible to elucidate the Shh-Ptc interactions through steric interference (2). The Cys-24, Asn-50, Asn-115, Ser-135, Ser-156, and Ser-177 residue sites experience significant loss of function as a result of attachment of a polyethylene glycol polymer with a molecular weight of approximately 5000 g/mol (PEG 5000), suggesting that these sites form or neighbor the surface of the Ptc binding site (2). Only Asn-50 and Ser-156 are susceptible to the addition of a pyrenyl moiety, which is significantly smaller than the PEG 5000 moiety, strongly suggesting that these specific residues form the binding site (2).

Shh-N possesses two alpha helices, nine beta sheets, and four random coils (2). No clear structural motifs are present. The aforementioned residues lie on the exterior of alpha helices and beta sheets which implicates their participation in Ptc binding as surface elements. Shh-N has an extended leading N-terminal tail and a compact globular core (2). 

Shh possesses a metal binding cleft responsible for coordinating a zinc (II) ion ligand (2). By the same steric interference techniques used to map the Ptc binding surface of Shh, Phe-30 and Pro-26 have been determined to be fundamental to the stability of the metal binding cleft by forming extensive hydrophobic contacts with aromatic residues neighboring the cleft, which include Phe-47, Trp-172, Tyr-174, and His-182 (2). Zinc (II) ion ligand coordination with other complexes is required for Shh-N-Ptc binding (7). The metal binding cleft provides stability necessary for the N-terminal to be extended remarkably in the tertiary conformation of Shh-N (2). This extension forms a leading N-terminal tail (2). The importance of the metal binding cleft in the tertiary conformation and Ptc binding of Shh-N also implicates the critical role of palmitoylation during dual lipid modification of the Shh-N (3, 7). 

The protein structures of the hedgehog protein family are highly conserved among species as well among the proteins themselves (2). Comparison of primary and tertiary protein structures of human Shh-N and similar tertiary structures to a query protein (9). A sum-of-pairs method is applied to comparison of intramolecular distances of a tertiary protein structure to determine similarity by evaluating a Z-score; Z-scores greater than 2 indicate significant similarity. The Dali server revealed a Z-score of 30.1 for the murine Shh-N monomer chain (9). This Z-score indicates a high degree of similarity between the 3D structures of human and murine Shh-N further corroborating the high degree of conservation observed in the hedgehog family.

While human and murine Shh-N have similar primary and tertiary protein structures, they differ significantly in certain secondary structure orientations. Murine Shh-N possesses two additional calcium ion ligand binding sites causing the globular core to open up to a greater degree than that of human Shh-N (10). According to the ExPASy isoelectric and molecular weight calculator, murine Shh-N has a mass of 18,997.34 Da and isoelectric point of 8.81 as opposed to the corresponding 19,683.16 Da and 8.81 (pI) of human Shh-N (5). Murine Shh-N is 7 residues shorter than human Shh-N (1, 10). The murine Shh-N lacks the leading N-terminal tail that human Shh-N possesses. The lack of a leading N-terminal tail allows for rotation of the metal binding cleft that may account for the opening of the hydrophobic core allowing for formation of two calcium ion ligand binding sites.