Smoothened_in_complex

Smoothened in complex with Vismodegib (PDB ID: 5L7I) from Homo sapiens is a trans-membrane protein that plays a key role in the Sonic Hedgehog (SHH) pathway (1, 2). The SHH pathway is important in tissue maintenance and re-growth in cells. While the SHH pathway is vital, its function and the way it works is not fully understood. Mutated Smoothened can lead to unregulated growth of cells and basal cell carcinoma (BCC), a form of cancer. Vismodegib is a synthetic tumor-suppressing drug commonly used to target the mutation of the Smoothened (3). Smoothened has a molecular weight of 141408.15 Da and an isoelectric point of 8.03 (4).

Smoothened is naturally found as a dimer, with two identical subunits. Each subunit is comprised of three domains, the extracellular cystein rich domain (CRD), a linker domain and a heptihelical trans-membrane domain (TMD) (2). The Smoothened, crosses the entire cell membrane, and is present in the extracellular membrane and intracellular spaces (2, 5). Smoothened is primarily composed of alpha-helices but beta sheets and random coils also contribute to the makeup of the protein (1, 6). Smoothened is classified as a G-protein-coupled receptor (GPCR) due to its structure and function because it passes through the entire membrane a total of seven times, and is responsible for signaling both outside and within the cell (7). Because GPCRs characteristically pass through the membrane a total of seven times, they are also referred to as seven-trans-membrane receptors (2, 8). GPCRs are a large class of proteins which have similar structural elements and functions. Smoothened only loosely fits into this family of proteins. Though it passes through the membrane seven times, it has many atypical characteristics for GCPRs. Smoothened is most uncharacteristic on the extracellular end, having a much more extensive loop structure, and stabilization through four disulfide bridges (6). Despite this, Smoothened is classified as a GCPR and is able to be comparable to many other GCPRs (8).

Like Smoothened in complex with Vismodegib, human P2Y12 receptor in complex with an antithrombotic drug (PDB ID: 4NTJ) from Homo sapiens is also a G-protein-coupled receptor (GPCR) (8). Smoothened and P2Y12 receptor are similar in both primary and tertiary structure. Primary structures are compared using an E-value based on Psi-Blast results (10). The E-value is 1.0E-12, indicating that the proteins have a similar primary structure. An E-value is considered significant if it is less than 0.05. E-values are calculated in Psi-Blast based on the Expect (E) value, which decreases at an exponential rate, as the sequence of two proteins is more similar. A value of 1 indicates one match in a sequence and therefore indicates little similarity. This calculation is impacted by the size of the protein (10). Tertiary structures are compared using a Z-score calculated using Dali Server (11). The Z-score is 22.6, indicating that the proteins have a similar tertiary structure. A Z-score is considered significant if it is greater than two. Dali-Sever calculates Z-scores based using a sum-of-pairs method based on intra-molecular distances (11). Both Smoothened and P2Y12 are made up primarily of alpha-helices. Both proteins are stabilized by cholesterol and are able to complex to synthetic drugs to regulate a cellular process. P2Y12, in complex with an antithrombotic drug, participates in the regulation of thrombus formation (8). As mentioned, Smoothened is an atypical GCPR, so while belonging to the same family and being statistically similar there are many differences between Smoothened in complex with Vismodegib and P2Y12 in complex with antithrombotic drug. Smoothened is a much larger protein with two identical subunits while P2Y12 is made up of only one subunit. The single subunit of P2Y12 has a shorter sequence than one of the two subunits of Smoothened (1). The molecular weight of P2Y12 is 53189.10 Da, compared to 141408.15 Da, the molecular weight of Smoothened (4). Smoothened in complex with Vismodegib is also more complex in structure and bonding, including more stabilization and extensive tertiary structure through disulfide bonding and interactions between specific residues in all three domains of the protein (2). Despite the fact the SHH pathway is not fully elucidated, it is apparent that Smoothened is more complex than P2Y12 in both structure and function (2, 8).

Smoothened in complex with Vismodegib has three domains. The linker domain, responsible for connecting the CRD and TMD, plays a minimal role in function of Smoothened (2). The linker domain does not contain any ligand binding sites or active sites. The linker domain does have a vital role in binding because it forms a small opening for the TMDs active site to remain in contact with the extracellular space (6). Several residues in the linker domain also appear to help stabilize the active site of the TMD when Vismodegib is present by contributing to an extensive hydrogen bond network. Arg-400 has the most stabilizing hydrogen bonding interaction, with evidence that His-470, Asp-473, Glu-518 and Asn-521 also contribute to hydrogen bonding (2, 6). Asp-473 is particularly important, as Vismodegib resistant basal cells carcinomas have been found to have a mutated Asp-473. As such, the linker domain plays a role in the operation of the heptihelical trans-membrane domain (TMD), the domain where Vismodegib binds.

TMD is found in the membrane, and crosses the membrane seven times, resulting in Smoothened being classified as a GCPR. Vismodegib is a TMD antagonist allowing it to be used as a cancer treatment (3). Vismodegib is stabilized when in complex with TMD by hydrogen bonding interactions from the linker protein. It is also stabilized by hydrophobic interactions from within the TMD (2). Vismodegib is partially composed of a methylsulfone, a non-polar group containing sulfur and oxygen atoms bound (3). Residue Phe-484 most extensively interacts with this group to stabilize and hold Vismodegib in the TMD active site (2, 3, 5). Non-polar residues, such as Phe-484, are likely to be found in the center of the TMD, as it is, to decrease interaction of non-polar side chains and polar solvent. The active site of the TMD also often contains sodium ions, which stabilize the polar molecules in the binding site. The true role of the sodium ions, which naturally occur in the body, is not fully known (8). Smoothened can have a varied TMD based on the binding in its active site. Vismodegib is only present in the body when introduced to treat basal cell carcinoma. Smoothened can present without a complex, no bound molecule in the TMD active site, or with other small molecules in complex in the active site (2). Smoothened in complex with cholesterol from Homo sapiens (PDB ID: 5L7D) is in complex with cholesterol in the CRD, but lacks a molecules in complex in the TMD (1). Smoothened can also be in complex with other anti tumor agents either natural or synthetic, represented by smoothened 7TM receptor in complex with an antitumor agent from Homo sapiens (PDB ID: 4JKV) and Smoothened Receptor structure in complex with cyclopamine from Homo sapiens (PDB ID: 4O9R). Cyclopamine is a natural tumor suppressor found in the human body (1). In all of these smoothened derivatives, the TMD is the only varied domain. In all species, the linker domain remains the same and the CRD is also consistent, in complex with cholesterol (1).

In Smoothened in complex with Vismodegib, the CRD is in complex with cholesterol and N-Acetyl-D-Glucosamine (NAG) ligand (2). The entire CRD is present in the extracellular space, and is primarily alpha-helices, but has more random coils than the TMD. The CRD gains stability from nine disulfide bridges. Four of these disulfide bridges play an essential role in forming the CRD binding pocket, or active site, where cholesterol is then able to bind (2, 5). These disulfide bridges also help bind the CRD and the linker domain, and result in hydrophobic interactions including residues Ile-185, Val-182, and Phe-187 (2). Cholesterol binds in the CRD and is help in the binding pocket by hydrogen binding (5). The hydrogen bonding present in human Smoothened is not known. In mice the hydrogen bonding occurs between cholesterol and residues Asp-99 and Tyr-134 (2). In humans, evidence suggests that Asp-95 also has hydrogen bonding interactions with the cholesterol ligand (2, 5, 13). The ligand, a prosthetic group, NAG, also adds another support to link the linker domain to the CRD. The CRD is not directly involved in tissue repair, which is the major function of Smoothened once activated. That being said, the presence of cholesterol in the CRD allows for signaling of the Smoothened in the Sonic Hedgehog (SHH) pathway (13).

The mechanism by which Smoothened is activated is still not fully understood (2). It is understood that once activated, Smoothened allows for unregulated cell growth (2). The interaction of the three domains are essential to understanding this activation, including the binding of cholesterol in the CRD active site. While this is the case, the mutation of nonpolar residues in the linker domain did not appear to have a significant impact on the binding of cholesterol, as was (2, 13). Further investigation of the interactions between the domains must be performed to have a better understanding of the activation of Smoothened. Furthermore, the understanding of the TMD active site must also be improved in the hopes to control basal cell carcinoma (BCC). While Vismodegib is often effective in the treatment of this cancer, more recently resistant forms have been seen clinically (3, 12). This complication adds another element of complexity to the understanding of the Smoothened activation and Sonic Hedgehog protein. More practically, this adds another level of complexity to the understanding of the treatment of basal cell carcinoma.