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Abstract

Proteins are inherently dynamic entities. While many functional aspects can be derived from their static or time- averaged structures, a comprehensive mechanistic understanding often requires knowledge about the specific dynamics underlying a biological process. NMR is a powerful technique to study these dynamics on different timescales ranging from picoseconds up to seconds. Different NMR methods can be exploited to investigate structural changes such as side chain rotations, molecular tumbling, folding and domain motions, which accompany binding events, allostery or catalysis. Dcp2 is a Nudix enzyme which catalyzes the removal of the 5' protecting cap structure from mRNA, resulting in an efficient termination of gene expression. The catalytic activity is already present in the isolated catalytic domain (CD), but it is incrementally increased by the N-terminal regulatory domain (NRD) and the activator proteins Dcp1 and Edc1. Although a number of crystal structures of Dcp2 decapping complexes has been solved, their biological relevance and the mode of activation remain controversial. We used TEMPO and ATCUN spin labels for paramagnetic relaxation enhancement (PRE) measurements on reporter methyl groups to elucidate domain orientations in the absence and presence of activators and substrate. The experimental data could not be fitted by a single spin label conformation, indicating that the spin label is flexible. To account for this, we generated an ensemble of 500 spin label conformations using a simulated annealing workflow in XPLOR-NIH. A combination of three spin label conformations was selected from this ensemble, based on a Monte Carlo approach implemented in Matlab, that minimized the difference between experimental and calculated PRE values. In agreement with published results 1 we find that the correlation between experimental and calculated PRE values does not increase with the use of more than three conformations. The procedure improves the identification of the actually sampled domain orientation in decapping complexes and thereby provides an accurate picture of the structure of the decapping complex in solution.