Hinge-bending domain movement of 5'-nucleotidase

We have determined structures of nine independent conformers using four different crystal forms. The relative orientation of the two domains of the protein (N-terminal domain in green and C-terminal domain in blue) differs by a rotation angle of up to 96°. Only in the closed conformation (right picture) the substrate is bound to the catalytic center, which is marked by the two metal ions shown in red. In the open conformation (left image) the substrate ATP is 25 Angstrom away from the active site.

The domain rotation observed for this enzyme differs markedly from other examples of domain movements in proteins. Here, the most common movement is a hinge-bending closure motion, by which the cleft between the two domains is opened and closed. In contrast, the rotation axis in 5'-NT (shown as a red arrow) passes close to the center of the C-terminal domain. As a result, this domain rotates approximately around its center. The residues at the domain interface slide along the interface, but the cleft between the two domains is not opened.

Left: Classical closure motion. Right: In 5'-NT the C-terminal domain rotates approximately around its center.

We are studying the domain rotation of this enzyme by site-directed mutagenesis, enzyme kinetics, X-ray crystallography and by other biophysical techniques using spectroscopic labels (e.g. for fluorescence spectroscopy) in solution.

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