1 Department of Physics and 3 Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Calcutta-700009, India
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Abstract |
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Keywords: glucocorticoid receptor/homology modelling/ligand-binding domain/steroid receptor complex/transactivation
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Introduction |
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The DBDs and LBDs for almost all the members of the steroid receptor superfamily are relatively well conserved, whereas the NTDs are less conserved (Hollenberg et al., 1985; Arriza et al., 1987
). The receptors have overlapping steroid-binding specificity as they bind to the same hormone-responsive elements in target genes (Arriza et al., 1987
; Pearce and Yamamoto, 1993
; Rupprecht et al., 1993b
). Mineralocorticoid receptor binds mineralocorticoids and most glucocorticoids with high affinity, whereas glucocorticoid receptor binds only glucocorticoids with high affinity (Arriza et al., 1987
, 1998; Rupprecht et al., 1993a
,b
). There is a functional similarity in both these classes of steroids, but glucocorticoids specifically regulate the development of specific tissues, glucose metabolism and the immune response (Miller and Tyrrell, 1995
).
A remarkable feature of the glucocorticoids is that they exert pronounced effects on a variety of tissues. In certain sites they stimulate the synthesis of macromolecular components, including nucleic acids and proteins. Sometimes the action of glucocorticoids is catabolic, resulting ultimately in cellular destruction (King, 1974). This diversity of action is reflected in the contemporary search for specific glucocorticoid-binding proteins in a wide range of experimental systems. Binding to the specific hormone receptors is acutely dependent on the presence of the 3-one A-ring and/or 11ß-hydroxyl group, which is the characteristic structural feature of all biologically active glucocorticoids. The 3-one A-ring of many progestins and corticoids is the area of greatest conformational flexibility in these molecules. This conformational flexibility exhibited by these naturally occurring hormones facilitates their highly specific interaction with a variety of proteins including synthesizing enzymes, transport proteins and target proteins (Duax and Norton, 1975
).
The theoretical model of mGR LBD was developed templating the X-ray structure of progesterone receptor (Williams and Siglar, 1998). The model receptor protein is then used to study the binding site interactions during complexation with cortisol and corticosterone by molecular modelling. The difference in accessible surface area (DASA) (Lee and Richards, 1971
) between mGR LBD and the ligand-bound protein is then calculated for both the steroidreceptor complexes. The interaction zone of mGR LBD with the steroid was revealed from this study. Binding of steroids to mGR LBD does not necessitate any structural change to the hydrophobic ligand-binding pocket. However, the modelling study did indicate a slight conformational change in the secondary structure on complexation of mGR LBD with both cortisol and corticosterone. This minor conformational change was found in the C-terminal site of the long helix H9 without any gross alteration of the ligand-binding pocket itself. This suggests that ligand binding may trigger a conformational modification which could account for the effect of ligand binding on transactivation by AF-2 (Bourguet et al., 1995
). Perhaps the ability of corticosteroids to stimulate the transactivation function of mGR depends on the stability of the steroidreceptor complexes (Hellal-Levy et al., 1999
).
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Materials and methods |
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The refined crystal structures of progesterone-bound LBD of the human progesterone receptor (hPR) (Williams and Siglar, 1998), LBD of estrogen receptor (ER) in complex with the endrogenous estrogen (Brzozowski et al., 1997
) and the LBD of human androgen receptor (hAR) in complex with metribolone (R1881) (Matias et al., 2000
) were taken from the Brookhaven Protein Data Bank (PDB entries 1A28, 1ERRand 1e3g, respectively) as starting materials. The amino acid sequences of three LBDs were extracted from these three crystal structures. The amino acid sequence of house mouse glucocorticoid receptor (mGR) (Danielsen et al., 1986
; Nohno et al., 1989
) was obtained from SWISSPROT Sequence Data Bank and was compared with the sequences of the crystal structures separately by pairwise sequence alignment using the software GAP (Needleman and Wunsch, 1970
) of the GCG package. The best similarity and identity of mGR with hPR LBD encouraged us to select the crystal structure of progesterone-bound LBD of hPR to develop a theoretical model of mGR LBD. A multiple sequence alignment among mGR, hPR, hAR and hER was done using the PILE UP (Feng and Doolittle, 1987
) program of the GCG package.
Coordinate assignment and minimization
The coordinates of mGR LBD were assigned by templating the X-ray structure of hPR LBD after aligning the two sequences as was observed in the GCG output. This coordinate assignment was done using the HOMOLOGY module (Biosym Technologies) of the InsightII program package. The model of mGR LBD was then put through energy minimization for 10 000 steps of the steepest descent method using the DISCOVER module of InsightII. The model was further subjected to energy minimization for 500 steps of the conjugate gradient technique that led to a refined structure of mGR LBD with an r.m.s. deviation of <0.001. The secondary structure prediction of mGR LBD was performed using the program DSSP (Kabsch and Sander, 1983), July 1995 version. The DISCOVER simulation package (Biosym Technologies) with the consistent valence force-field (Hagler et al., 1985
; Dauber-Osguthorpe et al., 1988
) was employed for minimization calculations.
Superposition and ligand docking
Using the Biopolymer module (Biosym Technologies) of InsightII, the refined model structure of mGR LBD was superposed on the crystal structure of progesterone-bound hPR LBD. The molecular structure of cortisol (Roberts et al., 1973) was then superposed on progesterone such that their steroid nuclei were practically coincident. The allocated cortisol could then be easily associated with the ligand-binding domain of the superposed glucocorticoid receptor. The hydroxyl group at C-17 of the cortisol in the mGR LBDcortisol complex thus prepared was deleted to mimic an mGR LBDcorticosterone complex. These two complexes were then subjected to energy minimization for 1000 steps of the conjugate gradient technique with the DISCOVER simulation package.
Solvent accessibility
The values of the accessible surface area for both the native protein (mGR LBD) and ligand-bound proteins were calculated using the HOMOLOGY module of InsightII. The differences in accessible surface areas between mGR LBD and ligand-bound protein were calculated for every residue. This DASA study traced the steroid protein interaction regions for the complexes of mGR LBD with both cortisol and corticosterone.
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Results and discussion |
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The postulated model of mGR LBD consists of 250 residues (Leu534Lys783), which folds into a hydrophobic ligand-binding pocket. The secondary structure of this model protein contains 11 helices, nine turns and four sheets. The pairs of helices H3, H4; H5, H6; and H10, H11 are almost contiguous pairwise. mGR LBD contains a relatively longer helix H9 (it contains 30 residues, whereas the PR contains 15, the RAR contains eight, the TR contains six and the ER contains nine). A shortening of the length of the helix H9 is observed in both the complexes of mGR LBD with cortisol and corticosterone. On complexation the helix H9 (718747) of the native protein (mGR LBD) becomes deformed and is shortened by one residue to become helix H9 (718746). This deformation at the C-terminal end of helix H9 is shown in Figure 3, where the trace of mGR LBDcortisol complex is superposed on the native protein. This superposition (Figure 3
) shows a perfect coincidence everywhere except from Ser740 to Asp748. The r.m.s. deviation in the aligned position is 0.147. The DASA study between mGR LBD and its complex with cortisol is represented by the bar graph in Figure 4(a)
. Figure 4(b)
represents the DASA study between mGR LBD and its corticosterone complex. The positive DASA values reveal the interaction zone of the receptor with its cognate ligands. The negative DASA values are found to be around the region of deformation arises from complexation. Figure 5(a)
represents the C
trace of mGR LBD, where H and S indicate
-helices and ß-sheets, respectively. Bound cortisol is shown in a space filling model. Figure 5(b)
represents the ribbon diagram of mGR LBD where the bound cortisol is shown in the space filling model.
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The hydrogen-bonding parameters associated with cortisol and corticosterone in complexation with mGR LBD are given in Table II. Table III
presents the binding energies of cortisol and corticosterone in their complexes with mGR LBD. Figure 6(a) and (b)
show a stereoscopic view of the binding site interactions of cortisol and corticosterone, respectively, with mGR LBD in a stick diagram. Final energies before complexation of mGR LBD and hPR LBD (X-ray structure) with steroids are given in Table IV
. Table V
represents the final energies of the receptors complexed to steroids. Tables IV and V
clearly reveal a high degree of agreement between the actual crystal structure and model structure, thereby confirming the reliability of the modelled steroidreceptor complex as well as the model of the uncomplexed mGR LBD.
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Notes |
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Acknowledgments |
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References |
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Received September 29, 2000; revised April 27, 2001; accepted April 30, 2001.