The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N {1H} nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 μM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 ∼ 0.9) and from the local correlation time <100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops β1/β2 and β5/β6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops β3/β4 and β6/β7, in contrast, is restricted, characterized by the values of order parameter S2 ∼ 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, τloc) when protein molecular mass (τc) increases. The use of negative values of the steady state 15N{1H} NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good agreement with the NMR data for the amide NH groups located in the protein core.