Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.     

Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain in complex with DNA and free in solution.

Molecular dynamics simulations have been performed on the glucocorticoid receptor DNA binding domain (GR DBD) in aqueous solution as a dimer in complex with DNA and as a free monomer. In the simulated complex, we find a slightly increased bending of the DNA helix axis compared with the crystal structure in the spacer region of DNA between the two half-sites that are recognized by GR DBD. The bend is mainly caused by an increased number of interactions between DNA and the N-terminal extended region of the sequence specifically bound monomer. The recognition helices of GR DBD are pulled further into the DNA major groove leading to a weakening of the intrahelical hydrogen bonds in the middle of the helices. Many ordered water molecules with long residence times are found at the intermolecular interfaces of the complex. The hydrogen-bonding networks (including water bridges) on either side of the DNA major groove involve residues that are highly conserved within the family of nuclear receptors. Very similar hydrogen-bonding networks are found in the estrogen receptor (ER) DBD in complex with DNA, which suggests that this is a common feature for proper positioning of the recognition helix in ER DBD and GR DBD.     

Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations.

Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between "open" and "closed" conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of this mutant were studied using15N spin relaxation experiments at three different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7x10(3)s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2x10(4)to 3x10(4)s-1at 291 K for a subset of residues. The temperature depen dence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.     

Conformational states of the glucocorticoid receptor DNA-binding domain from molecular dynamics simulations

Molecular dynamics simulations (MD) have been performed on variant crystal and NMR-derived structures of the glucocorticoid receptor DNA-binding domain (GR DBD). A loop region five residues long, the so-called D-box, exhibits significant flexibility, and transient perturbations of the tetrahedral geometry of two structurally important Cys4 zinc finger are seen, coupled to conformational changes in the D-box. In some cases, one of the Cys ligands to zinc exchanges with water, although no global distortion of the protein structure is observed. Thus, from MD simulation, dynamics of the D-box could partly be explained by solvent effects in conjunction with structural reformation of the zinc finger.     

Characterization of millisecond time-scale dynamics in the molten globule state of alpha-lactalbumin by NMR

The motional dynamics of the molten globule (MG) state of alpha-lactalbumin have been characterized using (15)N transverse relaxation rates (R2). A modified version of the Carr-Purcell-Meiboom-Gill (CPMG) R2 pulse sequence is proposed in order to overcome the loss of sensitivity that arises from extreme line broadening due to complex dynamics on the millisecond time-scale. Using this pulse sequence, chemical exchange rates were extracted by examining the (15)N transverse relaxation rates as a function of CPMG delay values. The results clearly illustrate that pervasive conformational exchange of 0.2-0.5 ms in the (15)N backbone resonances of the molten globule state of alpha-lactalbumin. The temperature dependence of the conformational exchange rates display standard Arrhenius kinetic behavior between 10 and 30 degrees C. Estimates of the activation energies range from 0.8 to 4. 4 kcal/mol, indicating a low energetic barrier to conformational fluctuations relative to native state proteins. The fluctuations and low energetic barriers may be critical for directing the search for contacts that will result in the transition from the MG state to the native state.     

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1–119) and a C-terminal DNA-binding domain (DBD, residues 130–236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein–DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.     

Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.     

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.