TY - JOUR
T1 - DNA-binding by the glucocorticoid receptor
T2 - A structural and functional analysis
AU - Dahlman-Wright, Karin
AU - Wright, Anthony
AU - Carlstedt-Duke, Jan
AU - Gustafsson, Jan Åke
N1 - Funding Information:
\[181\]T).h e ability of an appropriateloyr iented has contributetdo our understandinogf steroid ~t-helixt o recognizea specificD NA sequence receptoar ction.D omainso f the GR important was later clearly establishedfr om analysiso f for DNA-binding,s teroid-bindinng,u clearlo cal-protein-DNAc ocrystal\s[ 188-192H\]o. wevern, o ization, interaction with hsp90 and trans-structureo f a eukaryoticD NA-bindingp rotein activationh ave been identified.H owever,the had yet been reported. mechanismby which DNA-bounds teroidr ecep-We set out to determinteh et hree-dimensionatl ors affectt he rate of transcriptionainl itiation structureo f the GR DBD. This was particularly remainse lusive.G R can activatet ranscription intriguinga s it becamea pparentt hat several in yeastc ells, suggestingth at it acts via some proteins,w ith proposedr oles in development, fundamentalm echanismpreserved through shareda homologouDs NA-bindingmotif (for evolution\[ 1462, 68\].T hat is acts via a funda-a reviews eeE vans\ [33\]O). ncethe structureo f a mentafla ctori s also supportebdy the squelching proteini s known, it is importantth atsufficient (competitionfo r limiting transcriptionfa ctors) geneticaal nd biochemicadl ata is availablet o of basal transcriptiono bserveda s a conse-evaluateit s biologicasl ignificancae nd to allow quenceo f over-expressioonf a GR transactiva-modellingo f the interactiono f the proteinw ith tion domain\[ 180\]. Cell-free transcription possibleli gandsa ndt herefores,u chs tudiesw ere systemsa ble to respond to added hormone initiated. receptor\[ 152-156s\h] ouldf acilitatep rogressin To obtain sufficientq uantitieso f proteinf or this field. a detaileds tructuraal nalysist,h eGR DBD was expresseidn E. coli and the proteinp urifiedt o homogeneit\[y2 59\]I.n collaborationw ith two other groups,t he structureo f the proteinw as determinedu sing NMR-spectroscop\[y7 72, 67\]. We obtainede videncefo r the bindingo f a dimer of the GR DBD to a GRE and we identifieda segmenotf the proteini mportanfto r this dimer- ization\ [20,1 11,1421, 43\].W e initiated studies of specific amino acid-basep air interactions importantf or sequences pecificDNA recog- nition by the GR DBD \[266\]T. he resultsf rom theses tudiesa s well as a numbero f published reports\[ 747, 5, 112, 126, 127\] allowed us to proposea model of the GR DBD complex\[ 77\]. The structureo f the GR DBD represents only a first step towards an understandinogf GR DNA-binding.N ext it will be importantto determineth e structureo f the GR DBD-DNA complex.T his should be possibleu singNMR- spectroscopIyt. will also be importantto deter- mine the structureo f the full-lengthG R and its complexw ith DNA to investigatae possible contributionof otherd omainso f the proteinf or DNA-binding, either directly or more likely indirectly.U nfortunatelyt,h e GR is a rather large protein and attemptst o purify large quantitieso f the protein from prokaryotico r eukaryoticc ells have, so far, not been success- ful. Furthermoret,h e large size of the protein rules out the use of NMR-spectroscopfyo r a three-dimensionsatlr uctured eterminatioann d one can only hope that crystals suitable for X-ray diffractionc an be grown. During the courseo f theses tudies,t he work from a numbeor f laboratorieasr oundt heworld Acknowledgements--Twhiosr kwas supportebdy grants from the SwedishM edicalR esearcCh ouncil( No. 13X-2819),t he SwedishB oardf or TechnicaDl evelopment, KaroBioA B, andK arolinskain stitutetfos rskningsfonder.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1992/3
Y1 - 1992/3
N2 - The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors. This family includes, in addition to the receptors for steroid hormones, receptors for thyroid hormone, retinoic acid and 1,25-dihydroxy vitamin D3 as well as some receptors with as yet unknown ligands. The glucocorticoid receptor DNA-binding domain has been expressed in E. Coli. The purified protein binds to the same DNA sequences as the native receptor and is therefore suitable for biochemical and structural studies of the DNA-binding function of the receptor protein. This protein has been shown to bind as a dimer to its DNA-binding site. Protein-protein interactions facilitate DNA-binding and a segment responsible for these interactions has been identified close to the C-terminal zinc-binding site. The family of nuclear receptors, with their related DNA-binding sites, provides an opportunity to study determinants for DNA sequence recognition. A segment close to the N-terminal zinc ion has been shown to be responsible for the target specificity of glucocorticoid and estrogen receptors. DNA-binding domains of nuclear receptors include nine conserved cysteine residues which have been shown to coordinate two zinc ions and zinc has been shown to be required for the structural integrity and DNA-binding ability of the glucocorticoid receptor DNA-binding domain. A motif for DNA recognition, based around zinc ions, was first described for transcription factor IIIA and nuclear receptors were believed to recognize DNA via a similar motif. However, the three-dimensional structure determination of the glucocorticoid receptor DNA-binding domain shows that its structure is clearly different from that of the TFIIIA type zinc-binding domains.
AB - The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors. This family includes, in addition to the receptors for steroid hormones, receptors for thyroid hormone, retinoic acid and 1,25-dihydroxy vitamin D3 as well as some receptors with as yet unknown ligands. The glucocorticoid receptor DNA-binding domain has been expressed in E. Coli. The purified protein binds to the same DNA sequences as the native receptor and is therefore suitable for biochemical and structural studies of the DNA-binding function of the receptor protein. This protein has been shown to bind as a dimer to its DNA-binding site. Protein-protein interactions facilitate DNA-binding and a segment responsible for these interactions has been identified close to the C-terminal zinc-binding site. The family of nuclear receptors, with their related DNA-binding sites, provides an opportunity to study determinants for DNA sequence recognition. A segment close to the N-terminal zinc ion has been shown to be responsible for the target specificity of glucocorticoid and estrogen receptors. DNA-binding domains of nuclear receptors include nine conserved cysteine residues which have been shown to coordinate two zinc ions and zinc has been shown to be required for the structural integrity and DNA-binding ability of the glucocorticoid receptor DNA-binding domain. A motif for DNA recognition, based around zinc ions, was first described for transcription factor IIIA and nuclear receptors were believed to recognize DNA via a similar motif. However, the three-dimensional structure determination of the glucocorticoid receptor DNA-binding domain shows that its structure is clearly different from that of the TFIIIA type zinc-binding domains.
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U2 - 10.1016/0960-0760(92)90351-I
DO - 10.1016/0960-0760(92)90351-I
M3 - Article
C2 - 1562506
AN - SCOPUS:0026524233
VL - 41
SP - 249
EP - 272
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
SN - 0960-0760
IS - 3-8
ER -