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Vitamin D Receptor Home Page

Purpose and Program

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Sequence/Structure Alignment for NR-LBDs Used to Construct VDR-LBD Model I (14-Oct-99)

er  ----SLTADQ MVSALLDAEP PI------LY SEYDPTRPFS ---------- -----EASMM GLLTNLADRE LVHMINWAKR VPGFVDLTLH DQVHL 378

pr  -----QLIPP LINLLMSIEP DV------IY AGHDNTKPDT ---------- -----SSSLL TSLNQLGERQ LLSVVKWSKS LPGFRNLHID DQITL 750

vdr RPKLSEEQQR IIAILLDAHH KTYDPTYSDF CQFRPPVRVN D********* ****SQLSML PHLADLVSYS IQKVIGFAKM IPGFRDLTSE DQIVL 262

tr  RPEPTPEEWD LIHVATEAHR ST-NAQGSHW KQRRKFLPDD IGQSPIVSMP DGDKVDLEAF SEFTKIITPA ITRVVDFAKK LPMFSELPCE DQIIL 250

rar SYELSPQLEE LITKVSKAHQ ET----FPSL CQLGKYTTNS SADHRVQL-- -----DLGLW DKFSELATKC IIKIVEFAKR LPGFTGLSIA DQITL 262

rxr ----SANEDM PVERILEAEL AV----EPKT ETYVEANMGL NPSSP----- ------NDPV TNICQAADKQ LFTLVEWAKR IPHFSELPLD DQVIL 300
                 H1                   H2                                       H3                         H4


er  LECAWLEILM IGLVWRSMEH P--GKLLFAP NLLLDRNQGK CVEGMVEIFD MLLATSSRFR MM---NLQGE EFVCLKSIIL LNSGVYTFLS STLKS 468

pr  IQYSWMSLMV FGLGWRSYKH VSGQMLYFAP DLILNEQRMK ES-SFYSLCL TMWQIPQEFV KL---QVSQE EFLCMKVLLL LNTIPLE--- ----G 834

vdr LKSSAIEVIM LRSNESFTMD ----DMSWTC GNQDYKYRVS DVTKAGHSLE LIEPLIKFQV GLKKLNLHEE EHVLLMAICI VSPDRP---- ----G 345

tr  LKGCCMEIMS LRAAVRYDPE S--DTLTLSG EMAVKRKQLK NG-GLGVVSD AIFELGKSLS AF---NLDDT EVALLQAVLL MSTDRS---- ----G 331

rar LKAACLDILM LRICTRYTPE Q--DTMTFSD GLTLNRTQMH NA-GFGPLTD LVFAFAGQLL PL---EMDDT ETGLLSAICL ICGDRM---- ----D 343

rxr LRAGWNELLI ASFSHRSIAV K--DGILLAT GLHVHRNSAH SA-GVGAIFD RVLTELVSKM RD--MQMDKT ELGCLRAIVL FNPDSK---- ----G 382
      |        H5              S1       S2   H6               H7                    H8


er  LEEKDHIHRV LDKITDTLIH LMAKAGLTLQ QQHQRLAQLL LILSHIRHMS NKGMEHLYSM KCKN---VVP LYDLLLEMLD AHRLHA           551

pr  LRSQTQFEEM RSSYIRELIK AIGLRQKGVV SSSQRFYQLT KLLDNLHDLV KQLHLYCLNT FIQSRALSVE FPEMMSEVIA AQLPKILAGM VKPLL 929

vdr VQDAALIEAI QDRLSNTLQT YIRCRHPP-P GSHLLYAKMI QKLADLRSLN EEHSKQYRCL SFQPEC-SMK LTPLVLEVFG NEIS             427

tr  LLCVDKIEKS QEAYLLAFEH YVNHRKHN-- -IPHFWPKLL MKVTDLRMIG ACHASRFLHM KVECP--TEL FPPLFLEVFE DQEV             410

rar LEEPEKVDKL QEPLLEALRL YARRRRPS-- -QPYMFPRML MKITDLRGIS TKGAERAITL KMEI---PGP MPPLIREMLE NPEMFEDDS        426

rxr LSNPAEVEAL REKVYASLEA YCKHKYPE-- -QPGRFAKLL LRLPALRSIG LKCLEHLFFF KLIG---DTP IDTFLMEMLE APHQMT           462
                 H9                               H10       |     H11               H12


LEGEND: The sequence of human VDR (vdr) has been aligned with the sequences of five NRs of known structure human estrogen receptor (er), human progesterone receptor (pr), rat thyroid hormone receptor (tr), human retinoic acid receptor (rar) and human retinoate X receptor (rxr). The original multi-alignment was performed with PIMA, then adjusted manually, including inspection of the secondary structure alignments. Hyphens indcate gaps. Blue asterisks in the VDR sequence show the location of the large insertion (residues 162-221) which has not been included in the modeled structures. The VDR sequence shown begins at the start of the LBD (residue 121). Alpha helices are marked by a single underline and have been identified by the program DSSP except in the case of the thyroid hormone receptor whose coordinates are not publically available. Secondary structure boundaries in that case have been taken from Figure 1 in Wagner et al. (1995). Beta strands are marked in red and underlined and also identified by DSSP (except in the case of tr). Helices and strands are numbered according to the original scheme used for RXR (Bourguet et al., 1995). Residues involved in the secondary structural elements of VDR as predicted by model I are given in boldface for convenient reference. The PR sequence has been truncated four residues short of its C terminus to save space (it continues as ...FHKK).

HOMOLOGY MODEL OF VDR-LBD

General Considerations

X-ray crystallographic structures have been published for several nuclear receptor ligand-binding domains (NR-LBDs): (a) unliganded RXR (Bourguet et al., 1995), (b) liganded RAR (Renaud et al., 1995), (c) liganded TR (Wagner et al., 1995), (d) liganded ER (Brzozowski et al., 1997; Tanenbaum et al., 1998), (e) liganded PR (Williams & Sigler, 1998), and (f) liganded PPAR (Nolte et al., 1998). Sequence-homology comparisons indicate that TR would make the best template for homology-extension modeling of VDR, but the coordinates of this structure have never been released [not available in the Protein Data Bank (www.rcsb.org) as of 10/18/99]. We therefore selected PR as our principal template, but modeled two short segments on the corresponding parts of RAR (see below). We also excised 60 residues of the long insert in the VDR-LBD which projects outward between helices H2 and H3; secondary-structure predictions for this sequence are weak and there are no good matches to it in the PDB. Hence any attempt to model it would be arbitrary, and its position in the protein structure is such that it cannot play a significant role in ligand binding, coactivator binding or receptor dimerization (assuming that these functions are embodied in portions of VDR that are homolgous to those of other members of the NR family).

A substantial literature covers the topic of the conformation(s) accessible to 1,25-(OH)2-D3 and we thoroughly reviewed it before selecting a starting structure for the ligand. For the A-ring we selected the chair form with the C-1 hydroxyl group in an axial position (which makes the C-3 hydroxyl group equatorial). We tested both cis- and trans-conformations of the seco-B ring, normally preferring the latter. A standard steroid conformation was employed for the C,D-ring moiety.

Figure 1 depicts the sequence alignment used for model I; the legend describes its construction. Alignments were generated **before** deletion of the large inserted loop (positions 162-221). Note that a multi-alignment has much higher information content and reliability than a two-sequence comparison and our final manual adjustment took all six sequences into account.

Modeling Methods

We used various modules of the Insignt II software package from MSI to carry out the modeling calculation, first superposing all of the known NR-LBD structures to define the positions of helices and the shape of the ligand pocket with bound ligand. Residues 125-141 were chosen for H1, which is therefore longer than H1 of the PR template. Standard parameters were used to extend this alpha helix. The rest of the structure was built with the Insight II Homology Module using the coordinates of PR as a template and choosing the loops manually from the automatic selections generated by the software. In two parts of the sequence (residues 143-161 and 283-293) we used RAR coordinates in place of PR to give a "hybridcomplex" template. Considerations of accomodating the H2/H3 loop in an unstrained structure and positioning the beta-hairpin at the inner end of the binding pocket so as to account for affinity labeling and mutation data (see above) dictated the selection of the RAR segments in the model. All residue positions in the "hybridcomplex" were then mutated to the corresponding VDR amino acid sidechains.

We used the Builder Module of Insight II to construct the ligand molecule and then placed it in an initial docking position in the model based on the superposition of the ligands in the known liganded LBD structures and the result of affinity labeling studies with the 3-bromoacetate ester. Using the Docking Module we further explored ligand conformations and manually adjusted the 1,25-(OH)2-D3 position with reference to the bond and electrostatic energy terms. Next the liganded hybridcomplex was exported to XPLOR for further refinement. H-bond partners for the hydroxyl groups on the A ring were chosen on the basis of their position vis-a-vis the ligand as well as mutational data. Constraints were imposed so as to force the hydroxyl group on C-3 to form an H-bond to the sidechain of serine-237 and to force the C-1 hydroxyl to accept an H-bond from the guanidinium group of arginine-274 and donate one to the hydroxyl group of serine-275. Repeated rounds of minimization were conducted alternately with and without the H-bond constraint terms. The final structure generated was robust, changing very little when subjected to molecular dynamics. Note that throughout this procedure the sidechain of 1,25-(OH)2-D3 was **not** constrained to any particular position, but simply allowed to seek a minimum-energy position.

Truncation
Choice of template
Conformation of 1,25-(OH)2-D3
Sequence alignment
Insight II
Hybrid template
Ligand-docking
Modeling results
3° structure
ligand pocket
contacting residues
Comparison with experimental structures of liganded NRs



Sean Quinlan <wwwadmin@darwin.bu.edu>
last modified: Saturday, October 30, 1999 11:04:44 AM