Atomic resolution protein allostery from the multi-state structure of a PDZ domain.

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into structurally and dynamically correlated protein sites at atomic resolution. This is demonstrated in the present work for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery had been predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein, an allosteric conformational selection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by changing the distance between the ß-sheet 2, α-helix 2, and sidechains of residues Lys38 and Lys72. The observed induced fit-type apo-holo structural rearrangements are in line with the previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized and complex dynamic interplay of the PDZ2 domain owed by the structure-dynamics landscape.

The experimental accuracy of the uni-directional exact NOE.

We have established protocols to calculate exact NOEs (eNOE) from NOE data. eNOEs lend unprecedented precision to the calculation of distance restraints used for structure calculation. Moreover, as eNOEs are averaged quantities over all conformations of a molecule, they may contain accessible information of the sampled conformational space. In practice, a prerequisite for an exact interpretation is the evaluation of both NOESY cross-peak buildups. For large molecular sizes, the fraction of NOEs which can only be obtained from one cross peak typically increases. Distance restraints derived from such NOEs must be used with a tolerance for errors associated with the broken symmetry of the individual magnetization transfer pathways. The correct choice of upper and lower limits is particularly important for multiple-state ensemble calculation, where too narrow tolerances may lead to incorrect spatial sampling. In order to dissect these pathways in heavy-atom resolved 3D NOESY experiments, we analyze 2D [(1)H, (1)H]-NOESY experiments, which are the fundamental building blocks of the former. In combination with an analysis of excitation and inversion profiles of pulses on heavy atoms and relaxation effects during HXQC elements, we derive a rule for the correct choice of upper and lower distance limits derived from such uni-directional NOEs. We show that normalization of the cross- to the diagonal-peak intensities of the spins of magnetization destination rather than origin leads to similar errors of the distance restraints. This opens up the prospect of extended collection of unidirectional eNOEs.

Towards a true protein movie: a perspective on the potential impact of the ensemble-based structure determination using exact NOEs.

Confined by the Boltzmann distribution of the energies of the states, a multitude of structural states are inherent to biomolecules. For a detailed understanding of a protein's function, its entire structural landscape at atomic resolution and insight into the interconversion between all the structural states (i.e. dynamics) are required. Whereas dedicated trickery with NMR relaxation provides aspects of local dynamics, and 3D structure determination by NMR is well established, only recently have several attempts been made to formulate a more comprehensive description of the dynamics and the structural landscape of a protein. Here, a perspective is given on the use of exact NOEs (eNOEs) for the elucidation of structural ensembles of a protein describing the covered conformational space.