Biological function of biomolecules is accompanied by a wide range of motional behavior. Accurate modeling of dynamics by molecular dynamics (MD) computer simulations is therefore a useful approach toward the understanding of biomolecular function. NMR spin relaxation measurements provide rigorous benchmarks for assessing important aspects of MD simulations, such as the amount and time scales of conformational space sampling, which are intimately related to the underlying molecular mechanics force field. Until recently, most simulations produced trajectories that exhibited too much dynamics particularly in flexible loop regions. Recent modifications made to the backbone and ψ torsion angle potentials of the AMBER and CHARMM force fields indicate that these changes produce more realistic molecular dynamics behavior. To assess the consequences of these changes, we performed a series of 5-20 ns molecular dynamics trajectories of human ubiquitin using the AMBER99 and AMBER99SB force fields for different conditions and water models and compare the results with NMR experimental backbone N-H S 2 order parameters. A quantitative analysis of the trajectories shows significantly improved agreement with experimental NMR data for the AMBER99SB force field as compared to AMBER99. Because NMR spin relaxation data (T 1 , T 2 , NOE) reflect the combined effects of spatial and temporal fluctuations of bond vectors, it is found that comparison of experimental and back-calculated NMR spin-relaxation data provides a more objective way of assessing the quality of the trajectory than order parameters alone. Analysis of a key mobile -hairpin in ubiquitin demonstrates that the dynamics of mobile sites are not only reduced by the modified force field, but the extent of motional correlations between amino acids is also markedly diminished.

The oncoprotein MDM2 regulates the activity and stability of the tumor suppressor p53 through protein-protein interaction involving their N-terminal domains. The N-terminal lid of MDM2 has been implicated in p53 regulation; however, due to its flexible nature, limited data are available concerning its role in ligand binding. The quantitative dynamics study using NMR reported here shows, for the first time, that the lid in apo-MDM2 slowly interconverts between a "closed" state that is associated with the p53-binding cleft and an "open" state that is highly flexible. Our results reveal that apo-MDM2 predominantly populates the closed state, whereas the p53-bound MDM2 exclusively populates the open state. Unlike p53 binding, the small molecule MDM2 antagonist nutlin-3 binds to the cleft essentially without perturbing the closed lid state. The lid dynamics thereby represents a signature for the experimental and virtual screening of therapeutic antagonists that target the p53-MDM2 interaction.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with CaII binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for LnIIIs and YIII over CaII. Here we present the nuclear magnetic resonance solution structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein’s picomolar affinity for LnIIIs, and it suggests a role of unusual N i+1–H···N i hydrogen bonds, in which LanM’s unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM’s Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.