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 doma in (ER DBD) using NMR N-15 relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied pro tein fragments consist of residues Arg183-His267 of the human ER and residu es Lys438-Gln520 of the rat GR. The 15N longitudinal (R-1) and transverse ( R-2) relaxation rates and steady state {H-1}-N-15 nuclear Overhauser enhanc ements (NOEs) were measured at 30 degrees C at H-1 NMR frequencies of 500 a nd 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- t o nanosecond time-scales in two regions of the core DBD. These are the exte nded 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. Ln 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 R-2 as a function of the N-15 refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analy sis, we conclude that conformational exchange occurs in the loop of the fir st 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 local ized to two sites in the second zinc domain. The different dynamical featur es 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 conclud ed to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformation al dynamics on the micro- to millisecond timescales in the free state are i nvolved in intermolecular protein-DNA and protein-protein interactions in t he dimeric bound state. Based on the present data and the previously publis hed dynamical and DNA binding properties of a GR DBD triple mutant which re cognize an ER binding site on DNA, we argue that the free state dynamical p roperties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding. (C) 1999 Academic Press.