Low-Density Lipoprotein Receptor
Created by Emily Neal
Proteins play an integral role in maintaining the homeostatic environment within the human body. Low-density lipoprotein receptor(LDLR), a regulatory protein with a molecular weight and isoelectric point of 77622.64 Da and 4.66, respectively, maintains proper cholesterol levels within the cells of homo sapiens. (1) Lipoproteins transport lipids and cholesterol throughout the body via the circulatory system. Lipoproteins, such as LDL, bind at the extracellular ligand-binding domain of the 699 residue protein, LDLR. Once bound, the lipoprotein-LDLR complex enters the cell by endocytosis, where it assimilates into an endosome. In the low pH environment of the endosome, the lipoprotein dissociates from the LDLR and the LDLR recycles back to the cell membrane. (1,2) The LDL disassembles upon its arrival in the lysosome; this degradation process is the primary method of cholesterol uptake with the cell. (1) Mutations in the LDLR gene and ultimately the protein, can result in the development of familial hypercholesterolemia(FH). Patients with FH have an LDLR protein that ineffectively removes LDL from plasma, due to decreased receptor-ligand binding. (1) Elevated cholesterol levels within the body correlate with the development of atherosclerosis and coronary heart disease. (1,2)
The extracellular element of LDLR is classified by PDB code 1N7D. This extracellular component consists of two principle domains, N-terminal low-density lipoprotein (LDL) binding domain and EGF-beta-propeller domain. (1) However, these two domains do not encompass the entire LDLR protein. From the propeller domain, the LDLR protein connects to a domain of residues O-linked to oligosaccharides, followed by a transmembrane domain, and finally a domain including the C-terminal, located within the cytoplasm of the cell. Together these five domains recognize, bind, and invaginate LDL, allowing for the processing of the molecule within the cell, specifically within the endosome. The internalization of LDL, accompanied with cholesterol, allows for the incorporation of cholesterol into steroid hormones and cell membranes. (6)
The LDL binding domain allows for the attachment of LDL to the LDLR. This binding domain includes seven cysteine rich repeats of approximately 43 residues.(1) Each repeat, noted as R1-R7, contains three disulfide bonds and two loops.(1) The loops contains acidic residues, such as in R6 where D-235, E-237, D-239, D-245, E-246 form the calcium ion binding site. Calcium binds in R2-R7. Calcium binding is imperative as it allows for the binding of the LDLR to the ligand. (1) Calcium associates with the residues that create the binding pocket for LDL. Within these cysteine rich repeats are clusters of arginine and lysine residues, which increase the infinity of the LDLR for the ligand. R and K residues line the repeat's exterior facing the solvent; R-162 and R-164 line the exterior in repeat 4. Additionally within the cysteine rich repeats are negatively charged aspartates and glutamates. Specifically, in repeat 4, D-147, D-149, D-151, E-153, D-154, D157, E-158 form the binding location for LDL. The negative charge of the D and E residues attract to the positively charged residues of apolipoprotein-E, the LDL ligand, forming an electrostatic attraction between the two entities. (10) Repeats interact minimally with each other. The most significant interaction is the nonconserved linkages between each repeat, which is approximately five-residues in length, except for the twelve-residue connection between R4 and R5. (1) The repeat assembly lacks secondary structure, which allows for more flexibility within the protein.(1) This flexibility allows the protein to make slight structural modifications based upon molecules in close proximity and its surrounding environment. The structure of R1 could not be determined using x-ray diffraction due to the instability of the crystal structure. (1)
After the seven repeats, LDLR has two epidermal growth factor (EGF) subdomains (A, B), a beta-propeller, and another EGF subdomain (C). Similar to six of the repeats, subdomains A, B, and C have three disulfide bonds; however, only A and B have calcium ion binding sites.(1) The propeller is composed of six YWTD repeats, creating the six blades of the propeller. Each blade is an antiparallel beta-sheet of four strands, positioned radially around a central point. (2) The beta-propeller is essential to the LDLR, as it influences ligand binding within the endosome.
Research indicates that the propeller acts as an ligand at low pH values, allowing for the detachment of the LDL molecule from the LDL binding domain. This mechanism is thought to be activated by a change in pH. (1) The contact of R4 and R5 with the beta-propeller incorporates "hydrophobic and charged interactions," establishing a significant interface. (1) At an endosomic pH, residues Cys127-Cys163, in repeat 4, and Cys176-Cys210, in repeat R5, bind to residues Ile377-Gly642 of the beta-propeller instead of the lipoprotein ligand. (1) Specifically, hydrophobic interactions occur between Ala-120, Ile-130, Pro-141, Trp-144, and Asp-151 in R4 and the residues Trp-515, Thr-517, Trp-541, Lys-560 and His-586 in the beta-propeller. (1) Interactions between the repeats and the propeller are essential for proper function of the LDLR.
Additional structures encourage stability in the LDLR. For instance, three salt bridges are formed between R4 and the beta-propeller, specifically, one between Asp-147 and Lys-560. Tungsten molecules are located between R2 and R3 and between R3 and the beta-propeller, stabilizing the interaction between the entities. (1) Carbohydrate structures, such as N-acetyl-D-glucosamine attach to Asn135 and Asn251, also seem to influence the stability of the protein structure. (1) However, the functions of carbohydrates are not completely understood in the extracellular domain of LDLR. (1,9) Furthermore, subdomain EGF C connects to the beta-propeller at its base. The hydrophobic residues of EGF C (Trp-645, Leu-659, Leu-661, Pro-664, and Ile-666) are packed into the "loops of blades two and three" minimizing the interactions at the surface.(2) The propeller's YWTD repeat and the EGF complex are conserved across the family of lipoprotein receptors, indicating the necessity of these entities for cholesterol regulation within the body. (1)The inter and intra subdomain interactions, specifically between the EGF subdomains and beta-propeller, allow LDL to bind at a neutral pH and LDL to detach from the receptor at a low pH. (1, 7)
Altering any of the residues in the protein can significantly influence the structure and function of the protein. Specifically, if Gly-171, located in R5, becomes a valine, the hydrophobic environment changes. Research has revealed that the environmental change caused by one amino acid prevents LDL from attaching to the binding domain, suggesting that R5 has significant, if not the greatest influence on ligand binding. (8) Additionally, Gly-375, a residue between EGF B and the beta-propeller, creates a sharp kink in the protein structure allowing for the tight association of hydrophobic residues. If residue 375 is not glycine, then the structure of the protein is altered; the mutation is correlated with the development of familial hypercholesterolemia (FH). Currently, no medications on the market target the LDLR to treat FH.
Slight variations in protein structure have a tremendous impact on a protein's function as exemplified by the comparison of LDLR with proteins of similar structural characteristics. DALI, a search tool that determines the similarities between the tertiary structures of proteins, reveals a strong correlation between LDLR and nidogen, pdb 1NPE, and Brain Tumor (Brat) protein, pdb 1Q7F.(5) The similarity in structure between LDLR and nidogen results in a Z-score of 30.9 and rmsd of 2.0 Å, while Brat's z-score and rmsd are 22.0 and 2.8 Å, respectively. The root mean squared deviation (rmsd) indicates the distance between the backbones of superimposed proteins, the lower the value, the greater the structural resemblance. Likewise, the z-score indicates the degree of similarity between the tertiary structures, a score greater than 2.0 demonstrates significance.
The striking similarity between all three proteins lies with the inclusion of a beta-propeller into the structure. The beta-propeller has six blades, each a beta sheet.(2) The propeller influences binding and is conserved across several proteins.(3) In LDLR, the beta-propeller promotes the release of the lipoprotein at a low pH, by acting as an alternative substrate at the ligand-binding domain.(1) In contrast, the same structure in nidogen binds laminin, which promotes the organization of the basal laminae.(3) The beta-propeller of Brat has an electropositive top, which seeks the Pumilio puf domain resulting in the recruitment of the protein to the 3' untranslated region of the hunchback mRNA. The primary functions of the Brat protein is the regulation of brain size, cell size, and rRNA accumulation. (4) The amino acid sequence and structure of the beta-propeller are highly conserved across all the proteins because the beta strands have YWTD repeats. Additionally, both LDLR and nidogen have significant hydrophobic interactions necessary to stabilize the protein. When nidogen residues Asn 802 and Val804, contained within the propeller, are changed to Ser, the hydrophobicity changes resulting in the decreased affinity of nidogen for the ligand.(3) LDLR, nidogen, and Brat have similar tertiary structures and are important to ligand binding.