Conformational dynamics and molecular recognition: backbone dynamics of the estrogen receptor DNA-binding domain.
We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.
Wikström
A
A
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Berglund
H
H
Hambraeus
C
C
van den Berg
S
S
Härd
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016880:Anisotropy; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry*; D011965:Receptors, Glucocorticoid / Q000737:chemistry
D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid