1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.
Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.
Härd
T
T
Department of Chemistry, University of Utrecht, The Netherlands.
Kellenbach
E
E
Boelens
R
R
Kaptein
R
R
Dahlman
K
K
Carlstedt-Duke
J
J
Freedman
L P
LP
Maler
B A
BA
Hyde
E I
EI
Gustafsson
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D011965:Receptors, Glucocorticoid; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D006859:Hydrogen; D015032:Zinc