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Introduction

belongs to the albumin gene family. It is a multifunctional protein found in plasma, ascitic fluid, cerebrospinal fluid and other cell types as a surface protein. It is synthesized in the liver and is prevalent throughout the body. DBP is a major carrier of vitamin D3 and all of its metabolites. The active D3 hormone is critical for the maintenance of calcium levels, bone health, and regulates cell proliferation. This makes the D3 hormone of a compound of interest for many therapies, and by conjunction gives importance to DBP which can affect the pharmacokinetics of the D3 hormone. DBP ensures continuous metabolism of D3 hormone derived from human skin cells, and functions as storage for the hormone. Being part of the Human Serum Albumin family, it has similar structural components, however the unique interactions of DBP can be attributed to the arrangement of the helices of , shown in color. Since vitamin D3 analogs have so much potential as therapies, the understanding of DBP’s structure and binding properties could yield brand new in-sites into the workings of vitamin D3 pathways. This would allow the creation of new, more specific therapies centered around vitamin D3 metabolism.

Overall Structure

Basic Information

The secondary structure consists of mainly , which can be seen in pink. The quaternary structure of the protein consists of forming a complex. Due to “significant rotational freedom among the subdomains”[1] the two subunits when superimposed upon one another differ by about 6 degrees of rotation. The structure is about 52.1 kDA in size and made up of 458 amino acids.

Alpha Helical Domains

The Vitamin D binding protein consists of which are homologous to one another (Domain I: Blue, Domain II: Green, Domain III: Purple). containing 10 alpha helices, 9, and 4 being shorter than the other domains.

Vitamin D Binding Protein and Human Serum Albumin

The overall structure is closely related to that of the human serum albumin, to which it is homologous. The proteins are very similar yet the three dimensional structure differs somewhat to facilitate binding. The differences are due to bends at the C-terminal alpha-helices of the first and second domains in addition to rotations at the loops connecting the first two domains.

Actin Binding

The tertiary structure of the protein is optimized for it binding with actin, efficiently folding into a complex requiring little change of the structure. Once DPB binds to actin, the C-terminal alpha helix of the first domain and the loop between the second and third domain move to be in contact with the actin. The binding of actin to the differs from that of its homolog in the conformational changes that the protein undergoes, which can be attributed to differences in rotation in the first domain and the region between domain II III. Both green scenes are depicted with the same color scheme seen below.

Binding Interactions

The Vitamin D Binding Site

The Vitamin D binding site is located in domain I and contains helices 1 through 6. The binding site is lined with hydrophobic residues that interact with the hydrophobic parts of the vitamin D3 ligand.(locations highlighted in yellow) are formed between, the 25-hydroxyl and Tyr 32 with a distance of 2.85 angstroms, the 3-hydroxyl and Ser 76 with a distance of 3.01 angstroms and Met 109 with a distance of 3.01 angstroms.[1] Different analogs of the vitamin D3 ligand influence hydrogen bond locations and binding affinities. (yellow), for example, has a binding affinity to DBP of 1314, which is much greater compared to the affinity of 25OHD3, which is 667.[1] This is due to the and the aromatic residues of Phe 24, Tyr 34, Phe 36, and Tyr 38. The JX analog switches the meta hydroxyl group on JY to para, increasing the binding affinity to 2111. By switching the hydroxyl group to the para position tighter hydrogen bonds can be formed to the Ser 28 residue stabilizing the complex.[1]

Biological Relevance of The Vitamin D Binding Site

Vitamin D hormone 1,25(OH)2D3 used to treat renal osteodystrophy, hypoparathyroidism and osteoporosis. Administration of 1,25(OH)2D3 is limited due severe side effects, such as hypercalciuria, hypercalcemia and increased bone resorption.[1] The analogs of 1,25(OH)2D3 are being created to increase the activity and bind affinity without out the negative side effects.

Additional Features

Actin Binding Interactions

The most well documented auxiliary function of Vitamin D binding protein is its ability to bind and sequester circulating actin monomers. [4] The actin interaction occur at the sites shown in . While the vitamin D binding domain resides between leucine 35 and serine 49, the actin binding domain lies far away in sequence, between glycine 373 and glycine 403, the site is in the folded protein, with residues serine 42 and lysine 388 only 21.44Å apart on complementary subunits or 43.58Å apart on the same subunit.

At the level of the organism, this actin binding of DBP is an important mechanism for clearing actin from necrotic or apoptotic tissue [1]. This actin binding quality serves to prevent clotting and actin toxicity, as large quantities of circulating actin have been shown to be fatal to mice.

[4] Interestingly enough, DBP -/- mice are phenotypically normal and physically indistinguishable from normal mice, indicating that there may be other mechanisms and proteins involved in actin sequestration. [4]

Immune Function and Macrophage Activation

As the name suggests, macrophages (loosely translated from Greek as “big glutton”) are a class of white blood cells that move through the bloodstream and extracellular matrix in search of large debris and pathogens. They have been known to endocytose just about any foreign material that it does not recognize as self, including a wide range of microbes, cancer cells, and other debris. They are also important in antibody formation, as they often carry the instructions for how to destroy cells they have eaten.

DBP plays a role in macrophage activation through its conversion within immune cells to a compound called macrophage activating factor (MAF). At the site of the wound various immune cells act on DBP and deglycosylate it at several sites along the backbone. [6] This factor is important in the recruitment of macrophages to wound sites and other potential areas of infection. [6] Taking into account the above mention that DBP is important for clearing actin from wound sites, it makes logical sense that it would also be able to be easily converted to an endocrine or paracrine signal in this manner. [1]

Quiz Question 1

. is very similar to based on sequence similarity as well as a similar tertiary structure. The two proteins can both bind to actin, however HSA is unable to bind to Vitamin D3. Based on what you have learned about the binding nature DBP, and looking at the structures of the two proteins, hypothesize a reason why HSA cannot bind to Vitamin D3. How can altering only a couple of amino acids so greatly alter the function and tertiary structure of proteins?

See Also

• 1kxp
• 1kw2
• 1j7e
• 1j78
• 1ma9
• 1lot

Credits

Introduction - Uday Prakhya

Overall Structure - Elizabeth Swanson

Drug Binding Site - Alex Debreceni

Additional Features - Nick Rivelli

Quiz Question 1 - Robert Green

References

1. ↑ Verboven C, Rabijns A, De Maeyer M, Van Baelen H, Bouillon R, De Ranter C. A structural basis for the unique binding features of the human vitamin D-binding protein. Nat Struct Biol. 2002 Feb;9(2):131-6. PMID:11799400 doi:http://dx.doi.org/10.1038/nsb754

[1] Gomme PT, Bertolini J. 2004. Therapeutic potential of vitamin D-binding protein. Trends Biotechnol. 22:340–345.

[2] Haddad JG. 1995. Plasma vitamin D-binding protein (Gc-globulin): Multiple tasks. J. Steroid Biochem. Mol. Biol. 53:579–582.

[3] Otterbein LR, Cosio C, Graceffa P, Dominguez R. 2002. Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system. Proc. Natl. Acad. Sci. U. S. A. 99:8003–8008.

[4] Speeckaert M, Huang G, Delanghe JR, Taes YEC. 2006. Biological and clinical aspects of the vitamin D binding protein (Gc-globulin) and its polymorphism. Clin. Chim. Acta 372:33–42.

[5] Verboven C, Rabijns A, De Maeyer M, Van Baelen H, Bouillon R, De Ranter C. 2002. A structural basis for the unique binding features of the human vitamin D-binding protein. Nat. Struct. Biol. 9:131–6.

[6] White P, Cooke N. 2000. The multifunctional properties and characteristics of vitamin D-binding protein. Trends Endocrinol. Metab. 11:320–327.