Elucidating transient macromolecular interactions using paramagnetic relaxation enhancement

Abstract

Recent advances in the use of paramagnetic relaxation enhancement (PRE) in structure refinement and in the analysis of transient dynamic processes involved in macromolecular complex formation are presented. In the slow exchange regime, we show, using the SRY/DNA complex as an example, that the PRE provides a powerful tool that can lead to significant increases in the reliability and accuracy of NMR structure determinations. Refinement necessitates the use of an ensemble representation of the paramagnetic center and a model-free extension of the Solomon-Bloembergen equations. In the fast exchange regime, the PRE provides insight into dynamic processes and the existence of transient, low population intermediate species. The PRE allows one to characterize dynamic nonspecific binding of a protein to DNA; to directly demonstrate that the search process whereby a transcription factor locates its specific DNA target site involves both intramolecular (sliding) and intermolecular (hopping and intersegment transfer) translocation; and to detect and visualize the distribution of an ensemble of transient encounter complexes in protein-protein association.

Fig. 1

The intermolecular PRE in structure refinement of the SRY/DNA complex. (a) Oligonucleotides with location of the paramagnetic center (dT-EDTA-Mn²⁺) indicated by an asterisk and color-coded. The location of the specific SRY binding site is indicated by the solid bars and the site of intercalation of Ile13 is shown by an arrow. (b) Best-fit superposition of 40 simulated annealing structures (red) refined against 438 intermolecular ¹H-PRE restraints (31) on the restrained regularized mean coordinates (cyan) generated from structures based on NOE, dipolar coupling, J coupling and torsion angle restraints calculated without ¹H-PRE restraints (58). (c) Agreement between observed and calculated values of ¹H-Γ₂ rates for backbone and side-chain ¹H-PREs after refinement using a three-conformer ensemble representation for each paramagnetic center. Adapted from ref. .

Fig. 2

Impact of intermolecular PRE on coordinate accuracy of the SRY/DNA complex when only a single intermolecular NOE restraint located at the center of the protein-DNA interface is employed. Best-fit superposition of 30 simulated annealing structures (SRY, red, DNA, blue) calculated (a) without and (b) with 438 intermolecular ¹H-PRE restraints. The original restrained regularized mean structure of the SRY/DNA complex (58) determined using 168 intermolecular NOE restraints and 375 residual dipolar couplings is shown in cyan (SRY) and yellow (DNA). Adapted from ref. .

Fig. 3

Characterization of non-specific DNA binding of HMGB-1A by PRE. (a) Comparison of PRE profiles observed for the non-specific HMGB-1A/DNA complex and the specific SRY/DNA complex with two DNA duplexes bearing dT-EDTA-Mn²⁺ at opposite ends of the DNA. A diagrammatic depiction of the states giving rise to the observed PREs is shown on the right hand-side of the figure. (b) Semi-quantitative estimation of the distribution and occupancy of HMGB-1A binding sites along a 14-bp duplex DNA. There are 13 potential intercalation sites and HMGB-1A can bind in two orientations related by a 180° rotation (top left panel). The optimized distribution for the two orientations (bottom two panels) yields a PRE Q-factor of 0.36 (top right panel). Adapted from ref. .

Fig. 4

Intermolecular PRE in an exchanging system. (a) Diagrammatic depiction of a two site-exchange process involving major (99%) and minor (1%) species with paramagnetic-¹H distances of 30 and 8 Å, respectively. (b) Effect of increasing exchange rate on NMR line-shape with (red) and without (black) PRE. In the slow exchange regime the PRE is insensitive to presence of minor state; in the fast exchange regime, however, the PRE sensitive to the presence of minor species and can be used to reveal footprint of minor species. Adapted from ref. .

Fig. 5

Intermolecular PREs observed for the HoxD9/DNA complex in the slow (20 mM NaCl) and fast (160 mM NaCl) exchange regimes. (a) DNA duplex containing the HoxD9 specific binding site (boxed) and showing the location of the 4 sites used to introduce dT-EDTA-Mn²⁺ (one at a time). (b) Schematic illustration of the ground state specific complex and the target search process. (c) and (d) PRE profiles observed for site 1 at low (20 mM NaCl) and high (100 and 160 mM NaCl) salt, respectively. (e) and (f) Correlation between observed and calculated PREs for all 4 sites at low (20 mM NaCl) and high (160 mM NaCl) salt, respectively. Below the correlation diagrams, the PRE data are mapped on the structural model of the HoxD9/DNA complex, with the color scale depicting Γ₂ rates. Adapted from ref. .

Fig. 6

Intra- and intermolecular translocation in the HoxD9/DNA system. (a) PRE data were collected on HoxD9 in the presence of equal mixture of two DNA duplexes, one with and the other without the specific site (indicated in blue). In sample 1, the non-specific DNA bears the paramagnetic center and PREs only arise from intermolecular translocation; in sample 2, the specific DNA has the paramagnetic center and PREs can arise from both inter- and intra-molecular translocation. (b) Observed PRE profiles. (c) Schematic representation of sliding along the DNA with HoxD9 color coded according to the Γ₂(sample 2)/Γ₂(sample 1) ratio. Adapted from ref. .

Fig. 7

Intermolecular PREs for the EIN-HPr complex. EDTA-Mn²⁺ was conjugated to an engineered surface cysteine at 3 sites (E5C, E25C and E32C). (a) Correlation between observed and calculated intramolecular Γ ₂ rates for HPr. (b) Correlation between observed and calculated intermolecular Γ₂ rates measured on EIN and arising from paramagnetically labeled HPr. (c) Intermolecular PRE profiles observed for the 3 sites, with experimental Γ₂ rates denoted by the red circles, and the theoretical Γ₂ rates calculated from the structure of the stereospecific complex by the black line. Adapted from ref. .

Fig. 8

Ensemble refinement of intermolecular PRE data for the EIN-HPR complex. (a) The observed Γ₂ rates in the fast exchange regime are a weighted average of the Γ₂ rates for the specific complex and an encounter complex ensemble comprising N species. (b) Dependence of working (Q_e and Q_ee) and complete cross-validated (Q_free) Q-factors on ensemble size N. (c) Dependence of working Q factors on population of the encounter complex ensemble. (d) Correlation between observed and calculated Γ₂ rates obtained with a population of 10% for the encounter complex species represented by an ensemble of size N = 20. (Q_e and Q_ee are the average Q-factor <Q> for all 100 calculated ensembles, and Q_ee is the ensemble of ensembles average Q-factor. Adapted from ref. .

Fig. 9

Three views of a reweighted atomic probability map illustrating the distribution of HPr molecules on the surface of EIN that make up the ensemble of encounter complexes. The encounter complex probability map (green mesh plotted at a threshold of 20% maximum) is calculated from 100 independent calculations of ensemble size N = 20 at a population of 10%; the molecular surface of EIN is color coded by electrostatic potential (± 8 kT); and the location of HPr in the steterospecific complex is shown as a blue ribbon. Adapted from ref. .