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Two Key Proteins of the Vitamin D Endocrine System Come Into Crystal Clear Focus: Comparison of the X-ray Structures of the Nuclear Receptor for 1α,25(OH)2 Vitamin D3, the Plasma Vitamin D Binding Protein, and Their Ligands

2003; Oxford University Press; Volume: 18; Issue: 5 Linguagem: Inglês

10.1359/jbmr.2003.18.5.795

1523-4681

Mathew T. Mizwicki, Anthony W. Norman,

Biotin and Related Studies

Recently two breakthroughs have been achieved with respect to understanding the three-dimensional protein structures of both the vitamin D binding protein (DBP) and the nuclear receptor (VDR) for the steroid hormone 1α,25(OH)2-vitamin D3 [1α,25(OH)2D3] and the detailed shape of their respective bound ligands. The determination of the crystal structure of the ligand binding domain (LBD) of the VDR bound to its natural ligand, 1α,25(OH)2D3,1 and to several superagonist analogs of 1α,25(OH)2D32 was determined in the laboratory of Dino Moras in Strasbourg, France. Shortly thereafter, the determination of the crystal structure of DBP bound to either 25(OH)-vitamin D3 [25(OH)D3] or to a high-affinity analog was determined in the laboratory of Christel Verboven and C. De Ranter in Leuven, Belgium.3 The objective of this article is to compare the structural features of the VDR and DBP and their bound ligands, both with each other and with other related nuclear receptors or plasma steroid binding proteins. Identification of structural differences and similarities for these two proteins affords a unique insight at the molecular level into the functioning of two key components of the vitamin D endocrine system. Also, it is anticipated that this information will be useful from the perspective of drug development of new target organ selective analogs of 1α,25(OH)2D3. The vitamin D endocrine system is responsible for producing the key steroid hormone 1α,25(OH)2D3 and for its participation in the process of Ca2+ homeostasis and cell differentiation or inhibition of cell proliferation in selected target cells.4, 5 The integrated operation of the vitamin D endocrine system is dependent on two classes of proteins that each have inherent in their secondary and tertiary structure an LBD that allows the stereospecific binding of 1α,25(OH)2D3 and its synthetic analogs. These two key proteins are the nuclear receptor for 1α,25(OH)2D3, termed the VDR, and the plasma transport protein, the DBP. The LBDs of these proteins have neither primary amino acid sequence homology nor secondary or tertiary structural similarities with one another. Thus, it has been concluded that the VDR and DBP have distinct ligand-binding domains. A challenging structure-function problem has been to define the precise shape of the unusually conformationally flexible 1α,25(OH)2D3 and 25(OH)D3 (see below) as they function as the optimal ligands for the VDR and DBP, respectively. In addition, it has been an objective to understand the structural details that stabilize the interaction of each ligand with its cognate binding protein. The key organ in the vitamin D endocrine system is the kidney, where the renal proximal tubule is responsible for producing the hormonal 1α,25(OH)2D3 in accordance with strict physiological signals.6 The parent vitamin D3 is metabolized to 25(OH)D3 (by the liver) and then to 1α,25(OH)2D3 and 24R,25(OH)2D3 (by the endocrine gland, the kidney) as well as to 34 other metabolites.5 The seco-steroid 1α,25(OH)2D3 has been shown to initiate biological responses both through regulation of gene transcription as well as through rapid membrane-receptor-initiated pathways. From a nuclear receptor-gene transcription perspective, it is now clear that the vitamin D endocrine system embraces many more VDR containing target tissues than simply the traditional intestine, bone, and kidney. Notable additions to this list include the pancreas B-cell, placenta, activated B and T lymphocytes, monocytes, keratinocytes, and many cancer cells.5 The 1α,25(OH)2D3-mediated rapid responses can involve opening of voltage-gated Ca2+ channels, the rapid stimulation of intestinal Ca2+ absorption known as transcaltachia, or the activation of phosphatidyl-3′-kinase.7 Vitamin D3 and all its daughter metabolites, including 1α,25(OH)2D3, are unusually conformationally flexible (Fig. 1). Three key aspects of the 1α,25(OH)2D3 molecule confer a unique range of conformational mobility on this molecule. (1) The intact eight carbon side-chain of vitamin D and related seco-steroids can easily assume numerous shapes and positions in three-dimensional space by virtue of rotation about its five carbon-carbon single bonds (see Fig. 1B). A discussion of the consequences of the side-chain conformational mobility has been previously presented.8, 9 (2) The cyclohexane-like A-ring is free to rapidly interchange (many thousands of times per second) between a pair of chair-chair conformers (see Fig. 1C); this has the consequence of changing the orientation of the key 1α and 3β hydroxyls between either an equatorial or an axial orientation.10 (3) In the seco B-ring (see Fig. 1D), 360° rotational freedom about the 6-7 carbon-carbon bond of the seco B-ring allows conformations ranging from the more steroid-like 6-s-cis conformation to the open and extended 6-s-trans form of the hormone.8 It is generally accepted that this conformational mobility of vitamin D seco-steroids is displayed by the molecules in both an organic solvent as well as an aqueous environment similar to that encountered in biological systems. Thus, the VDR receptor for 1α,25(OH)2D3 and DBP must accommodate a highly conformationally flexible ligand. Conformational flexibility of vitamin D using 1α,25(OH)2D3 as an example. (A) Structure of 1α,25(OH)2D3 indicating the three features of the molecule that confer unusual (in relation to other steroids) conformational flexibility on the molecule. (B) For the side-chain, a dynamic 360° rotation around the five carbon-carbon single bonds as indicated by curved arrows. The dots indicate the position in three-dimensional space of the 25-OH group for 384 readily identifiable side-chain conformers that have been determined from energy minimization calculations.8 The left structure depicts the CD-ring and side-chain viewed from the top of the molecule. The middle view shows the same top view with the addition of dots depicting the position of the 25-OH in the energy minimized conformers. An in-plane view or C/D rings “edge-on” view of the position of the 25-OH group is depicted in the right structure. (C) The rapid (thousands of times per second) chair↔chair interconversion of the A-ring of the seco steroid that generates the two distinct chair conformers: the α-chair (C19 methylene “down,” the 1α-OH axial and the 3β-OH equatorial) and the β-chair (C19 methylene “up,” the 1α-OH equatorial, and the 3β-OH axial). (D) The rapid (thousands of times per second) 360° rotational freedom around the 6,7 single bond of the seco-B ring, which allows conformations ranging from the more steroid-like (6-s-cis) conformation to the open and extended (6-s-trans) conformation (see also Fig. 5). Based on evaluation of the human genome database, it is known that there are a total of 48 members of the nuclear receptor superfamily.11 In this superfamily, 28 receptors have an LBD that binds a small ligand molecule; of these 28 receptors, 8 are classical hormone receptors and 20 orphan receptors.11 The VDR belongs to the subfamily of nuclear receptors with an LBD that binds a classical hormone; this includes (as ligands) the glucocorticoids (GR), progesterone (PR), estrogens (ER), aldosterone (MR for mineralocorticoid), androgens (AR), thyroid hormone (TR), the hormonal forms of vitamin A (RAR), and the VDR.12, 13 Comparative studies of the VDR with all the steroid, retinoid, and thyroid receptors reveal that they have a common structural organization consisting of six domains14 with significant amino acid sequence homologies across all the domains (see Fig. 2A). The different domains act as distinct functional modules that can function independently of each other.15 Schematic models of the VDR and DBP. (A) The VDR is comprised of 427 amino acid residues that are divided into six domains (A-F). The numbers below the VDR indicate the amino acid residue boundaries for the various domains. The VDR belongs to a superfamily of nuclear receptors that have the same general A-F domain organization. The C domain, the most highly conserved, which contains the DNA binding domain, defines the superfamily; it contains two zinc finger motifs. The E domain or ligand binding domain (LBD) is less conserved and is responsible for binding 1α,25(OH)2D3 or its analogs and transcriptional activation. The A/B domain of the VDR is much smaller than other members of the superfamily. The portion of the intact VDR that was crystallized and subjected to X-ray crystallographic analysis included residues 118-427, but with deletion of the “loop” region of the hinge domain D, specifically residues 165-215 (see text). (B) DBP consists of 458 amino acid residues and is divided into three domains (I, II, and III). The numbers below the DBP indicate the amino acid residue boundaries for the various domains. Domains I, II, and III have been postulated to have evolved from a progenitor that arose from the triple repeat of a 192 amino acid sequence.43 However, domain III is significantly truncated at the C terminus. The 25(OH)D3 binding cleft is associated with the first six α-helices or residues 1-110 of domain I. The actin binding property of DBP is associated with a portion of domains I and III that clamp the actin while it rests on domain II. The DNA binding domain, C, is the most conserved domain throughout the family. About 70 amino acids fold into two zinc finger-like motifs. Conserved cysteines coordinate a zinc ion in a tetrahedral arrangement. The first finger, which contains four cysteines and several hydrophobic amino acids, determines the hormone response element (HRE) specificity; the HREs are comprised of specific nucleotide sequence that are located in the promoter of the gene to be regulated by the receptor(s) cognate ligand.15, 16 The second zinc finger, which contains five cysteines and many basic amino acids, is also necessary for DNA binding and is involved in receptor dimerization.17, 18 The zinc fingers identify the receptors cognate HRE and physically interact with the HRE to form a receptor + ligand + HRE-DNA complex. The next most conserved region is the steroid binding domain (region E). This region contains a hydrophobic pocket for ligand binding and also contains signals for several other functions, including dimerization,19 nuclear translocation, and hormone-dependent transcriptional activation.15-17 The A/B domain, which is quite small in the VDR (25 amino acids), is also known as the immuno- or transactivation domain. This region is poorly conserved in amino acid number and size, and its function has not been clearly defined. An independent transcriptional activation function domain is located within the A/B region,12, 17 which is constitutive in receptor constructs lacking the ligand binding domain (region E). The relative importance of the transcriptional activation by this domain depends on the receptor, the context of the target gene promoter and the target cell-type.20 Domain D is the hinge region between the DNA binding domain and the ligand-binding domain. The hinge domain must be conformationally flexible to allow the DNA binding and ligand binding domains some flexibility. The VDR hinge region contains 65 amino acids and has immunogenic properties. A dramatic advance in understanding of the three-dimensional structure of the LBD of steroid receptors has occurred over the past 7 years with the X-ray crystallographic structure determination of the LBD of five steroid hormone-related receptors. These include the LBDs of the TR, RAR, ER, PR, and the poroxisome proliferator-activated receptor γ (PPARγ); this work has been reviewed by Weatherman et al.21 Also, an X-ray structure is available for the LBD of the unoccupied 9-cis retinoic acid receptor retinoid X receptor (RXR).19 Furthermore, ER LBD X-ray structures are known for a ligand (raloxifene) that can act as an antagonist of the transcriptional activation function of the nuclear receptor.22 Very recently, the crystal structure of a modified version of the ligand binding domain of the nuclear receptor for vitamin D, bound to 1α,25(OH)2D3, has been determined at a 1.8-Å resolution.1 A follow-up X-ray crystallographic report compared the VDR LBD and bound ligand for 1α,25(OH)2D3 with that of four superagonist analogs of 1α,25(OH)2D3.2 The structure of the LBD of the human VDRnuc spans amino acid residues 143-427, [COOH terminus] but without residues 165-215, which were in an “undefined” loop in the hinge region of domain D (see Figs. 2A and 3A). In comparison with other nuclear receptors, the length of the VDR loop region is significantly longer. The removal of the flexible insertion domain in the VDR LBD produced a more soluble protein that was more amenable to crystallization. The VDR LBD protein structure is very similar to the LBD of the five other X-ray crystallographic-determined nuclear hormone receptor structures.21 The VDR LBD structure consists of a three-stranded β-sheet and 12 α-helices that are arranged to create a three-layer sandwich that completely encompasses the ligand 1α,25(OH)2D3 in a hydrophobic core (see Figs. 3A and 3B). Impressively, all six X-ray structures are so similar that their ribbon diagrams are virtually superimposable indicating a remarkable spatial conservation of the secondary and tertiary structures.21 In addition, the AF-2 domain of the C-terminal helix 12 (domain F; residues 404-427), contributes to the hormone binding pocket suggesting that the presence of a ligand could play a role in receptor activation. (A-C) Three dimensional structure of the VDR ligand binding domain for residues 118-425 (Δ165-215 is the loop deletion) with its bound ligand as determined via X-ray crystallography.1 (A) Ribbon structure of the VDR LBD bound to 1α,25(OH)2D3 as its ligand. The same orientation of the VDR LBD is presented in A-C, where each of the 12 helices have the same color designation. Each of the 12 helices (e.g., helix 1 [H1]) can be identified by individual colors (as well as the labels on A). The colors of the individual helices are as follows: H1, green; H2, gray; H3, light blue; H4, purple; H5, yellow; H6/β-sheet, pink; H7, red; H8, aqua; H9, bronze; H10/H11, gold; H12, black; Δ165-215 (loop deletion), rust. The white regions represent loops and other flexible regions of the receptor molecule. The ligand 1α,25(OH)2D3 has its atoms colored so that oxygen is red and carbon blue. (B) The VDR LBD is shown as a Corey-Pauling-Koltun (CPK) space filling model. The position of helix-12 (black) in the “closed” position1 effectively sequesters the ligand from the external environment of the protein, indicated by the absence of visible carbon and oxygen atoms from 1α,25(OH)2D3 in this view. (C) The VDR LBD is shown in stick representation with the important amino acid residues that stabilize the ligand binding to the LBD being highlighted as gray stick structures. Each gray stick structure is identified with a label specifying a particular amino acid that participates in noncovalent binding interactions with the ligand 1α,25(OH)2D3. 1α,25(OH)2D3 is colored as in Fig. 1A. S237 of H3 (light blue) forms a H-bond with the 1α-OH group of the ligand. R274 of H5 (yellow) further stabilizes the 1α-OH group by formation of a second H-bond. Y143 of H1 (green) forms a H-bond with the 3β-OH group of the ligand. The 3β-OH group also forms a second hydrogen bond with S278, located at the C-terminal end of the white loop region between H5 and H6 (see A). The 25-OH group of the ligand forms two hydrogen bonds with the LBD: one with H305 (C-terminal end of loop connecting H6 and H7, white) and the second with H397 of H10/H11 (gold). W286 of H6/β-sheet (pink) stabilizes ligand binding by forming π-π orbital overlaps with the ligands triene system (see Fig. 1A, seco B ring). V418 of H12 (black) makes van der Waals contact with C26 of the ligand, which aids in anchoring H12 in the closed position. (D-F) Three-dimensional structure of DBP for residues 1-458 as determined by X-ray crystallography.3 The X-ray structure of DBP was determined separately with two different ligands. These ligands were 25(OH)D3 and 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor vitamin D3 (analog JY); their structures are shown in Figs. 3G and 5. (D) Illustrates the three domains (I, yellow; II, light blue; III, green) of the DBP in a ribbon structure representation. The atoms of the ligand, 25(OH)D3 are colored so that oxygen is red and carbon blue. The ligand binding domain of the DBP is a crevice located on the surface of domain I. (E) This illustrates the CPK space filling structure of DBP, with white regions indicating flexible regions of the molecule. Virtually the entire top, surface (β-face) of the 25(OH)D3 (blue and red) is exposed to the external environment. (F) The ligand binding cleft (helices 1-6) and important residues (gray stick structure, labeled) known to stabilize and thought to interfere with binding of vitamin D sterols are depicted. The natural ligand 25(OH)D3 is shown here in the binding cleft and forms a H-bond with S76 (3β-OH) and Y32 (25OH). Also shown are analog JY's two H-bonding partners, M107 (3β-OH) and E15 (side-chain OH), and the two residues that are in close enough proximity to the phenol ring of JY and therefore thought to make favorable π-π orbital overlaps with the analog (F24 and Y68). V51, like V418 of the VDR LBD, helps stabilize ligand binding by forming van der Wall's interactions with methyl groups of the side-chain. F36 and S79 are residues implicated in either hindering or stabilizing binding of analogs of the two major vitamin D metabolites (see text). (G) Conformation of the optimal ligands for the VDR and DBP as determined by X-ray crystallography of the ligand bound to its cognate protein. The ligands have their atoms colored so that carbon is blue, hydrogen is gray, and oxygen is red; also, each carbon-19 methylene group (see Fig. 1A) is colored green as a “marker” for orientation of the A-ring. (G, Top) The shape of 1α,25(OH)2D3 as a ligand in the VDR LBD, in a stick (left) or CPK space filling (right) rendition, is shown as a twisted or bowl-shaped 6-s-trans orientation. The A-ring is in the β-chair conformation (see Fig. 1C) and the side-chain is oriented “northeasterly” at 2 o'clock as defined by its global energy minimum (see Fig. 1B “dot map” with line tracing of the side-chain.8 (G, Middle) The shape of 25(OH)D3 as a ligand for DBP in a stick (left) and CPK space filling (right) model, is shown as a hook. The A-ring is in the α-chair conformation (see Fig. 1C), and the side-chain is oriented behind and nearly perpendicular to the CD-ring. (G, Bottom) The shape of 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor vitamin D3 (analog JY) as a ligand for DBP is shown in the same manner as 25(OH)D3; its traditional structure is given in Fig. 5. The functional groups of the amino acids comprising the interior surface of the VDR ligand cavity interact very selectively with the ligand 1α,25(OH)2D3 to form a stable ligand-LBD complex (Fig. 3C). 1α,25(OH)2D3 is positioned in the LBD so that the A-ring enters the binding cavity first and then moves to the deep interior of the receptor so that the trailing side-chain and the 25-hydroxyl group are just “inside” the LBD, which is “closed” as a consequence of helix 12 moving from its “open” position (characteristic of the unoccupied VDR LBD). The 1α-hydroxyl participates in two hydrogen bonds with Ser237 (helix-3) and Arg274 (helix-5). The 3β-hydroxyl group participates in two hydrogen bonds with Ser278 (helix-5) and Tyr143 (helix-2). The 25-hydroxyl group is hydrogen bonded to both His305 (Loop helices 6/7) and His397 (helix-11). The C6-C7 single bond exhibits a trans conformation that deviates by 30° from the planar extended form of the ligand (see Fig. 4). Illustration of the orientation of the 6,7 carbon-carbon single bond of the optimal ligands for VDR [1α,25(OH)2D3], and DBP [25(OH)D3 and JY]. The circle illustrates the 360° rotation around the 6,7 single bond of 1α,25(OH)2D3 (see Fig. 1D). At 0° the planar 6-s-cis and at 180° the planar 6-s-trans conformations are shown. The intermediate α-triene and β-triene conformations are shown at 90° and 270°, respectively. The arrows indicate the angle of rotation about the 6,7 axis for the DBP ligands 25(OH)D3 (149°), JY (148°), and the VDR ligand 1α,25(OH)2D3 (211°). It has been generally assumed for receptor-ligand interactions that the ligand is frozen in a single conformation dictated by both the structural constraints of the ligand and the three-dimensional architecture of the peptide chains that create the LBD of the receptor(s). The ligands for the RAR and TR, as for the VDR, are all conformationally flexible, and the X-ray crystallographic structure for each receptor indicated that only one definitive conformer was present in their ligand binding domain.23, 24 This clearly show that the capability of steroid receptors to capture one ligand conformation from a large population of available flexible conformers is a general feature of several proteins. Among the 1000 or more analogs of 1α,25(OH)2D3 that have been prepared,5 two classes of analogs have attracted interest: those that have an inverted stereochemistry at carbon-20 of the side-chain (e.g., analog IE; see Fig. 5) and those which have two complete side-chains attached to carbon-20 (e.g., KH; see Fig. 5). The 20-epi analogs are significantly more potent (300-1000×) than 1α,25(OH)2D3 with respect to cell differentiation or inhibition of cell proliferation and have been termed “superagonists.”25 The ability of the 20-epi analogs, when bound to the VDR, to effect conformational changes in the VDR as judged by different protease sensitivity patterns correlates with the efficiency of these analog-receptor complexes to interact with coactivators and modulate gene transcription.26 It was proposed that the 20-epi analogs when bound to the VDR induced a different conformation from that of 1α,25(OH)2D3 that is more resistant to protease digestion, an observation that is consistent with its greater potency in promoting gene transcription. However, when the X-ray crystal structures of the VDR bound separately to two 20-epi analogs were determined, the VDR conformations were identical.2 The fact that the X-ray structure is static and therefore doesn't completely represent the dynamics of the protein in solution can provide one explanation for the differences in protease sensitivity patterns, but the overall shape and major stabilizing interactions made by the ligand would be expected to remain similar to allow for the receptor to form proper surface requirements needed to interact with coactivators.27 In addition, the expected trypsin site, which could generate the altered protease pattern, is located at the C terminus of helix-11, near the deleted loop of the VDR X-ray construct. A local altered conformation or unwinding at this site could likely be stabilized by more energetically favorable ligand-LBD contacts formed by the modified ligand, thereby rendering it accessible to the protease. Another possible explanation for the potency of these superagonist analogs is that the activity displayed is not achieved by selective protein conformational changes, but perhaps by differences in pharmacologic properties, such as a longer half-life. A similar conclusion had also been reached for the retinoic acid receptor, where numerous complexes with two natural ligands and several analogs demonstrated that the ligand did not mediate changes in the shape of the ligand binding pocket.28 Or said differently, it is the conformationally flexible ligand that adapts to the constraints of the LBD of the receptor. Structure of analogs of 1α,25(OH)2D3 and 25(OH)D3 that are optimal ligands for either the VDR or DBP. Ligands for the VDR and ligands for the DBP. The chemical name of each analog and its properties are presented in Table 2. The analog KH, also known as Gemini, has two side-chains (one 20-normal and one 20-epi side chain), and therefore is unusually bulky in relation to 1α,25(OH)2D3. KH is also an effective ligand for the VDR,29 binding 40% as 1α,25(OH)2D3, but is 13-fold more potent than 1α,25(OH)2D3 with respect to transactivation of the osteocalcin promoter. It will be interesting to learn whether it is the bulky ligand side-chain or the VDR LBD that accommodates to achieve a highly functional receptor-ligand complex. The vitamin D-binding protein (DBP), also known as group-specific component (GC-globulin),30, 31 is the serum protein that serves as the transporter and reservoir for the principal vitamin D metabolites throughout the vitamin D endocrine system.32, 33 These include 25(OH)D3, the major circulating metabolite (KD ∼ 6 × 10−9 M),34 and the steroid hormone 1α,25(OH)2D3 (KD ∼ 6 × 10−8 M). DBP can be up to 5% glycosylated and is known to be one of the most polymorphic proteins, with three common allelic variants and over 124 rare variants known.35 DBP's plasma concentration (4-8 μM) is approximately 20-fold higher than that of the total circulating vitamin D metabolites (∼107 M). DBP binds 88% of the total serum 25(OH)D3 and 85% of serum 1,25(OH)2D3, yet only 5% of the total circulating DBP actually carries vitamin D metabolites.36 In addition to the vitamin D metabolite binding properties of DBP, the protein has been shown to function as a high-affinity plasma actin-monomer scavenger, functioning in concert with the protein gelsolin to prevent arterial congestion.37 There are stoichiometric, 1:1, amounts of DBP and actin in their high affinity heterodimer; the actin/DBP's KD is ∼10−9 M. The X-ray crystallographic structure of the DBP-actin complex has been recently determined38, 39 but is not considered in detail here. DBP has been proposed to be involved in the transport of fatty acids40; its KD for fatty acids is ∼10−6 M. Also, DBP has also been implicated in playing a role in complement C5a-mediated chemotaxis41 and has been found to be associated with immunoglobulin surface receptors on lymphocytes, monocytes, and neutrophils.42 DBP (∼53 kDa) is a member of the albumin multi-gene family of proteins consisting of albumin (human serum albumin [HSA]),43 α-fetoprotein (AFP), and α-albumin/afamin (AFM).44 AFP (∼70 kDa) has an analogous function to albumin in the fetus and is measured clinically to diagnose or monitor fetal distress or fetal abnormalities, some liver disorders, and some cancers; however AFP has no known function in adults. To date, no known biological functions for AFM have been demonstrated.44 HSA is the major protein component in human plasma and binds a number of relatively insoluble endogenous compounds, including fatty acids, bilirubin, and bile acids.45 The known multi-functionality of DBP [both vitamin D metabolite and actin binding] separates it from the other members of its family and other steroid transport proteins like the retinal-binding protein (RBP) and thyroid binding globulin (TBG). However, two proteins that bind and transport sterols, sex hormone binding globulin (SHBG) and uteroglobulin (UG), have been implicated in other physiological functions other than steroid transport. SHBG, which binds sex steroids in blood, triggers cyclic adenosine monophosphate (cAMP)-dependent signaling through binding to specific cell surface receptors in prostate46 and breast cancer cells.47 The X-ray structure of SHBG has been determined48; SHBG crystallized as a homodimer. In each SHBG monomer, the steroid ligand intercalates into a hydrophobic pocket within a β-sheet sandwich.48 UG, which binds progesterone, displays potent anti-inflammatory and immunomodulatory properties.49 The three domain structures of DBP are shown in Fig. 2B. Domains I, II, and III have been postulated to have evolved from a progenitor that arose from the triple repeat of a 192 amino acid sequence43; however, domain III is significantly truncated at the C terminus. The position of the vitamin D metabolite and actin binding domains are specified in domains I and portions of domains I, II, and III, respectively. The two members of the albumin protein family that have had their X-ray crystallographic structure determined are DBP3, 38 and HSA50, 51; besides sharing extensive primary amino acid sequence homology, they unsurprisingly have the same secondary structure containing similar three-domain partitioning and α-helical folds.52 Physiologically DBP and HSA are functionally alike in that they bind fatty acids, but as with apparently all other protein families, slight sequence changes in the genes of these two proteins leads to small perturbations in the proteins' tertiary structure, leading to their dramatically different physiological functions. The X-ray structure of the thyroxine binding globulin (TBG) is known.53 TBG is the major plasma transport protein for iodothyronines; however, there is no sequence or structural homology between DBP and TBG. The X-ray crystallographic structures of the human DBP with a bound ligand of either 25(OH)D3 or 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor vitamin D3 (analog JY) has been recently determined3; the structures of 25(OH)D3 and JY are given in Fig. 3G. Both crystals contained two nonidentical DBP molecules present in the asymmetric unit.3 The only major difference between the two structures is that both DBP molecules are occupied in the JY structure, while only one is occupied in the 25(OH)D3 structure. 25(OH)D3 was absent in the two recent X-ray crystal structures of DBP complexed with an actin monomer.38, 54 In the DBP:actin complex, the actin is bound in a groove formed by the three domains, where domain I and III flank the actin monomer as it sits on top of domain II. The comparison of the X-ray crystal structures of HSA and DBP establishes that the overall topologies of DBP and HSA are similar50, 55 (Fig. 2B), but the detailed structural orientation of the three dom