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Abstract:
Enzymes are dynamic molecular machines. Many insights into the molecular details of their function have been
gained from crystal structures. But in the case of highly dynamic enzymes crystal structures are prone to packing
artifacts. They also hide dynamic information that is often crucial for the understanding of the enzymatic function. A
striking example is the bilobed decapping enzyme Dcp2. It catalyzes the removal of the protecting 5’ cap from
eukaryotic mRNAs and thereby regulates gene expression. The activity of the C-terminal catalytic domain (CD) of
Dcp2 is increased in a stepwise manner by the N-terminal regulatory domain (RD) and the activator proteins Dcp1 and
Edc1. The two domains of Dcp2 are connected by a flexible linker and Dcp2 has been shown to be highly dynamic.
Several crystal structures of Dcp2 in the free state and in complex with these activator proteins have been solved.
These structures can be divided into six groups with vastly different domain orientations. Due to the dynamic nature of
Dcp2 it is challenging to determine whether the obtained crystal structures are adopted in solution. This explains why
the mechanisms of activation and the structure of the catalytically active state of the enzyme still remain controversial
despite this wealth of structural information. To address these questions we explore the conformations that Dcp2
samples in solution using a suite of methyl TROSY based NMR experiments. By combining CSP, 13
C-CPMG
relaxation dispersion, NOESY and PRE experiments we show that Dcp2 samples three different structural states in
solution: an open and a closed conformation and a catalytically active form. The apo and the activator bound enzyme
complexes exchanges between catalytically impaired open and closed conformations. Substrate binding to the
Dcp1:Dcp2 complex competes with the closed conformation and results in a highly dynamic assembly. The stable
catalytically active state of the decapping complex is only formed in the presence of substrate and both activators,
which is explained by a novel crystal structure of the quaternary complex. In summary, we provide a detailed model of
how the conformational landscape of Dcp2 is modulated by decapping activators and how this increases the catalytic
activity.
