Parathyroid Hormone-Related Peptide Receptor
Created by Jesse Howell
The parathyroid hormone-related peptide receptor (3c4m) is found in Homo sapiens. The protein, abbreviated PTH1R, is a B G protein-coupled receptor. This receptor contains an N-terminus extracellular domain, meaning that it can protrude of out the cell membrane, and has a stable structure held together by three disulfide bonds. As with most G protein-coupled receptors, PTH1R has a C-terminal domain with seven transmembrane helices (1). PTH1R complexes in the binding pocket with either PTH or PTHrP to perform two independent functions. The protein is hard to crystallize and is ,therefore, often bound to a maltose binding protein to lower the solubility thus increasing the susceptibility of crystallization (1).This protein receptor is important for regulating and maintaining bone structure through calcium and phosphorus homeostasis, and any irregularities in this function provides severe complications including dwarfism and tumors (4).The molecular weight and isoelectric point (pI) for the protein are 62854.3 Da and 5.84, respectively.
PTH1R is a membrane protein that is made up of 2 identical 539 amino acid polypeptide chains as shown in the subunit structure (1). PTH1R has two primary functions depending on if it is activated by PTH or PTHrP. When activated by PTH, the protein controls calcium and phosphorus levels in the body and when activated by PTHrP, the protein controls bone formation and bone elongation. In the body 2gh9, the liver and bones have the highest concentrations of PTH1R. Mutations in the protein or its binding mechanism lead to dwarfism and several defects in calcium and phosphorus levels in the body. Defects can also lead to a potentially fatal disorder in which several tumors appear on the bones in the body. Most mutations are rare and the treatment is relatively unknown. Medical breakthroughs with this protein allow easier treatment of osteoporosis and the diagnosing and discovery of dwarfism (1).
The secondary structure of the protein is important to its function. It is made up of two identical chains. The protein polypeptide chains are composed of 35% alpha helices, 20 helices altogether with 190 residues. The chains are also composed of 17% beta sheets; there are 33 strands and 95 residues (1). The remainder of the protein is made up of random coils. The secondary structure of the entire molecule is made up of a three-layer, hot dog-like shape. The triple-layer structure has the dimensions 40 Å x 25 Å x 10 Å. The top layer of the PTH1R is primarily formed by the N-terminal of the ECD in the shape of a single longer α-helix. The second layer of the structure is broken up into two pieces: left and right. On the left is a β-strand hairpin and the right side of the layer is formed by two beta strands: β3 and β4. The third and final layer of the protein is formed by several β-strands and a loop connecting β4 to α2. All three layers of the protein are held together by three disulfide bonds (1). The disulfide bonds are located between several Cysteines. The cysteine-cysteine disulfide bonds lie between Cys-170 and Cys-131, Cys-148 and Cys-108, and Cys-117 and Cys-48. These three bonds and the triple-layer structure the hold together reveals an overall stable and well-defined protein. All of these structural features allow for very high ligand binding activity (8). The secondary structure of the ECD itself consists of two α-helices and four β-strands (1).
There are two main conformations of PTH1R and those conformations are dependent on which peptide chain, PTH or PTHrP, the PTH1R is bound to. PTH is the parathyroid hormone. PTH is an endocrine hormone, made up of an 84 amino acid polypeptide that activates PTH1R to regulate calcium and phosphorus homeostasis. The receptor interaction correlates directly with the phosphorus and calcium levels in most mammals (7). PTH docks as an amphipathic α-helix that docks into the hydrophobic groove of the ECD domain. The PTH helix creates a hot dog-like structure, where the PTH is the hot dog, and the two outer layers of the ECD make the bun. In the triple layer ECD, a β-hairpin and extended β turn make up the middle layer; the top ridge is formed by the N-terminal helix, while the C-terminal helix creates the bottom ridge. Hydrophobic interactions dominate the binding process. In this structure, the PTH α-helix is anti-parallel to the C-terminal helix and the N-terminal helix is oriented toward the transmembrane domain. The N-terminal residues of the PTH are directly involved in activating the PTH1R. The hydrophobic face of the PTH that packs tightly to the hydrophobic groove is made up of residues Val-21, Trp-23, Leu-24, Leu-28, Val-31, and Phe-34 (1).
The Hydrogen bonds in the binding mechanism of PTH to PTH1R strengthen the complex. The C-terminal group that was amidated forms a hydrogen bond with the backbone amide of Thr-163 and the carbonyl of Thr-162. This amidated group also stabilizes the helix by capping the backbone carbonyl Val-31 of PTH. These particular hydrogen bonds show that the amidated group is important to the receptor-PTH complex, and is thus important for the function of the protein. Asp-30 of PTH1R caps the N-terminal backbone amide of PTH and forms a hydrogen bond with the side chain Asn-16 of PTH. Arg-20 of PTH forms hydrogen bonds with the backbone carbonyls of both Asp-29 and Met-32 of PTH1R. The hydrogen bond interactions of the PTH-PTH1R complex are inconsistent with the other B GCPR complexes where the residues that come into contact with Arg-20 are not conserved. Arg-20 is important to determine the ligand specificity. It is postulated that this is one key as to why the PTH and PTHrP activations of PTH1R are similar in structure, but different in function (1).
The second conformation involves the parathyroid hormone-related protein. PTHrP is a 141 amino acid polypeptide that controls tissue development upon activation of PTH1R. The PTH1R ECD forms a hydrophobic groove. The PTHrP ligand forms an amphipathic helix and then occupies the groove. In this step, high electron density is observed for all PTHrP residues between 13 and 34. Phe-23, Leu-24, and Ile-28 form the intermolecular interface with the groove of the ECD that is formed by Leu-41, Ile-115, Ile-135, Phe-138, and Tyr-167. The peptide N- and C- termini are both anchored by several hydrogen bonds. The conserved Arg-20 has several interactions with different residues, including a salt bridge to Asp-137, an intermolecular hydrogen bond to the backbone carbonyl of Met-32, and an intramolecular hydrogen bond with the side chain of Asp-17. After residue Ile-31, the helix begins to unwind at the C-terminus allowing more hydrogen bonds to form (7).
The hydrogen bonds in the receptor-PTHrP complex include a δ-nitrogen of His-32 and the backbone amide nitrogen of Tyr-167.Hydrogen bonds are formed between the side chain hydroxyl group of Thr-33 and both the backbone amide nitrogen of Ala-165 and the backbone carbonyl of Thr-163. The backbone carbonyl of Ala-165 also forms hydrogen bonds with the amide nitrogen of THR-33 and Ala-34. An additional salt bridge is formed between Arg-19 and Glu-35. The salt bridge is likely a result of crystal packing interactions. This more anabolic interaction shows promise for tissue development data with less chances of creating side effects involving hyperkalemia (7).
Both perform their functions by activation of the PTH1R, but they have entirely separate functions. In both chains, the first 34 residue segments have a high binding affinity for PTH1R. Segments 1-14, which share 8 amino acid sequence identities, interact with the 7 trans-membrane helical domains, while segments 15-34, which only share 3 amino acid identities, interact with the N-terminal ECD. The crystalline structure of the either PTH or PTHrP binds to the extracellular domain of the PTH1R. Both will bind to the ECD as an amphipathic α-helix to a hydrophobic groove in the ECD. Where the PTH binds to the ECD in a straight helical formation, the PTHrP helix unwinds with respect to the C-terminal residues Ile-15 and Ile-31, and curves slightly. Each of the polypeptide chains are bound to the hydrophobic face in the ECD, and are then attached, as if by an anchor, by Arg-20 and Leu-24. Both interactions have coupling between Leu-41 and Asn-23, and Ile-115 and Asn-27 (7).
Because of differences at positions 23, 27, 28, and 31 of each of the peptides, the PTHrP-ECD complex is fitted more loosely, while PTH buries more surface area, becoming more tightly packed. The packing differences allude to the functional differences of the complexes. Although both of these methods involve binding in the same way to the ECD, the receptor will shift the side chain conformations of either Leu-41 or Ile-115 to allow for the difference. In the PTH-bound structure, the receptor accommodates the larger side chain of Trp-23 by adopting the less favorable X1=trans, X2=gauche (-) rotomer. This allows the indole ring of Trp-23 to stay in contact with the δ2 methyl group of Leu-41. PTHrP binds differently by adopting the favored X1=gauche (+), X2=trans rotomer. This enables the δ1 methyl group of Leu-41 to maintain contact with the phenyl ring of Phe-23. The Leu-41 acts as a switch for either the PTH or PTHrP sequences and the Ile-115 is shifted to allow for the curvature of the PTHrP helix. Understanding these shifting methods is important for the development of different therapeutic treatments for osteoporosis (7).
When the PTH1R ECD is purified, it binds to PTH and to PTHrP in a 1:1 monomeric complex with the ligand, as explained above. This is consistent with most class B GPCR ECD-ligand complexes. However, if the crystalline structure lacks a ligand with which to bind, it will dimerize using the same mechanism as either PTH or PTHrP recptor interaction. The PTH1R ECD binds my means of mimicking the previously described ligand mechanism: The C-terminal of the ECD will form an α-helix and, just as with either PTH or PTHrP, this helix will insert itself into the binding grove. The helix has an anti-parallel arrangement that constrains the possible orientations of the dimer. The structure of the dimer involves a 90o turn between the first trasnmembrane helix and the α-helix. Neither the C-terminal tail nor the 7 transmembrane helix contribute to the structure or function of the dimer (4).
The helix binding to the groove primarily mediates the dimerization, but there are several hydrogen bonds that occur in the dimerization mechanism. Asn-176 forms a hydrogen bond with Arg-179. Hydrophobic interactions with the binding groove of the ECD are caused by hydrogen bonding between the base of the groove with Val-183 and Leu-187. Ile-115 makes hydrophobic contact with the aliphatic portion of Arg-186. The polar, negatively charged Glu-182 carboxylate forms a hydrogen bond with the guanidino group of Arg-186. Ag-186 then forms a hydrogen bond with the backbone carbonyl of His-114. The binding groove in the ECD has a different surface topology than in the PTH or PTHrP complexes because of a 1-1.5Å lateral shift in residues 172-174. This difference allows Phe-184 to come into hydrophobic contact with the groove. Little is known as to why or what function this dimerization serves, but with research, a better understand may lead to further understanding or even categorization of class b GPCRs (4).
A comparable protein to PTH1R is the maltose/maltodexrin-binding protein (2gh9) which is found in thermos thermophiles, a gram negative eubacterium. The protein is made up of one single 386 amino acid polypeptide chain. This differs from PTH1R, which is made up for two polypeptide chains. A superimposition of the two proteins show similarity in structure. MBP are used as receptors for transport processes involving bacteria, as well as, neurotransmissions. Structurally, the protein is made of 2 loops and 3 helical segments with several hydrogen bonds causing steric blocking (6).
The secondary structures of the two proteins are compared; there are similarities in that the secondary structure of 2gh9 also containa alpha helices, beta sheets, and random coils. The proteins contain the maltose biding fold formed by 2 separate domains connected by β-sheets. Each of the domains are made of a α-β-α sandwich, similar to the α-β-βα of the PTH1R. A hinge of 2 antiparallel β-strands connects the 6 β-strand amino terminal and 5 β-strand carboxy terminal. Another structural similarity is that the binding site of the protein is within the hydrophobic center of the domain. When the domain is open, the hinge is loose; a ligand will insert itself into the fold and become complexed with the hydrophobic groove through 9 hydrogen bonds. This differs from the binding of the PTH1R only in that it is amino acids that connect the ligands to the binding site (6). The MBP has a Z-score of 45 (11). The Z-score is used to find proteins with similar tertiary structure. The score of 45 means that the tertiary structures are similar. MBP BLAST search E-value was 9e-37.The E-value is used to find proteins with primary structures similar to that of the protein of interest. The E-value for MBP is less than .05 and is therefore significant. The sequences of the proteins are similar, but not identical (6).
PTH1R is involved in skeletal growth when complexed with PTHrP. Ollier disease is a nonhereditary disease in which benign tumors appear on the bones of humans. In the growth plate, there is an indian hedgehog homolog, known as IHH, and PTHrP loop. When the IHH binds to its receptor, patched, the result is a PTHrP. This then complexes with the PTH1R causing upregulation. This binding then affects the pace of the chondrocyte differentiation by delaying them. The delay causes irregularities in bone growth. This entire process is begun because Arg-150, which is large and basic, is changed into a cysteine, which is medium and polar. Using this knowledge of the effects of the amino acid change, research was begun to discover other mutations affecting human bone growth (3).
Certain dangerous natural defects in PTH1R can lead to Blomstrand chondrodysplasia, a lethal endocchronal bone maturation. This defect is caused by a mutation in which Pro-132 is replaced by a leucine in the N-terminal receptor. Both the original amino acid and the replacement amino acid are medium sized and hydrophobic. The discovery of this defect is easily tracked and signaled throughout development. Further insights into this discovery show why the receptor-ligand relationship is important and what structural features are going to be needed for the receptors to function normally (9).
Mutations in the receptor-ligand complex can result in Jansen’s metaphseal chondrodysplasia, which is a condition caused by hypercalcemia. The PTH1R mediates the osteoblast activity which, when activated by PTHrP, slows the differentiation of chondrocytes, which then affect normal bone growth. Without a perfect combination of the necessary amino acids, the bones will either become dwarfed or elongated (5). JMC affects the growth plate and often leads to short limb dwarfism. The first mutation, named H223R, involves the second transmembrane domain where a conserved isoleucine, medium sized and hydrophobic, residue at location 458 is changed to an arginine, which is large and basic. This mutation, although rare, is the most frequently seen mutation, with 8 known mutants to date. The second mutation, known as T410P, is located in the sixth membrane domain and involves the very same change of the adenine to the arginine. The T410P mutant eventually developed a PTH dependence in their receptor which matches the affinity of a wild-type of the receptor. This mutation is extremely rare and has only ever been identified in one patient (2).