The crystal structure of 5'-nucleotidase (5'-NT) from E. coli, also known as UDP-sugar hydrolase, has been determined using four different crystal forms at 1.7 Å, 2.2 Å, 2.1 Å and 1.9 Å resolution. 5'-NT is monomeric and consists of two domains connected by an a
-helix. The major difference between the four crystal structures is a hinge-bending domain movement of 96° which divides the protein structures in an open and closed conformation. Two endogenous zinc ions in the active site of the open conformations are coordinated in a trigonal-bipyramidal way by protein ligands and a metal-bridging water molecule or a (bi)carbonate ion. In the closed conformation two manganese ions in the active site are coordinated octahedral by protein ligands, phosphate or inhibitor molecule, a metal-bridging and a monodentate binding water molecule. The active site shows structural homology to related enzymes of the superfamily of b
a
b
a
b
-metallophosphoesterases, including Ser/Thr protein phosphatases and purple acid phosphatases, but differs in the metal coordination sphere of the site 1 metal ion and other active site residues. The most prominent difference is the presence of a second domain in 5'-NT that forms part of the active site, located in a cleft between the two domains.
In the open conformation, substrate ATP is bound to the C-terminal domain at a distance of about 20 Å from the catalytic center. In the closed conformation, cocrystal structures of 5'-NT in complex with the products adenosine and phosphate and complexed with the substrate analogue inhibitor a
,b
-methylene-ADP have been determined in order to study the reaction mechanism.
E. coli 5'-NT exhibits a unique 96° domain motion in which the smaller C-terminal domain rotates approximately around its center such that the residues at the domain interface move predominantly in the direction of the interface. This movement differs from a classical hinge-bending closure motion which involves an opening of the substrate or ligand binding cleft between two domains such that the residues of the cleft move predominantly perpendicular to the domain interface. Structures of the open and closed forms with substrates and inhibitors show that the substrate moves by 20 Å with the large domain rotation into the catalytic site. Nine independent conformers have been analysed in the four crystal forms. The domain motions derived from a comparison of these conformations show that all conformational changes can be described as domain rotations around axes that are roughly located in one plane. This plane includes the domain centers and the hinge. Two residues, Lys-355 and Gly-356, form the core of the hinge region and undergo a large change of the main-chain torsion angles.
