Abstract
The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.
Highlights
THE JOURNAL OF BIOLOGICAL CHEMISTRYVol 276, No 43, Issue of October 26, pp. 40274 –40281, 2001 Printed in U.S.A. Unusual Conformational Changes in 6-Hydroxymethyl-7,8dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR*
The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-Å resolution with a crystallographic R factor of 0.191
Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK1⁄7MgAMPPCP
Summary
Vol 276, No 43, Issue of October 26, pp. 40274 –40281, 2001 Printed in U.S.A. Unusual Conformational Changes in 6-Hydroxymethyl-7,8dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR*. The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-Å resolution with a crystallographic R factor of 0.191. Comparative analysis of the crystal structures of the apoHPPK and its ternary complex has revealed the interactions of the enzyme with the substrates at the atomic resolution and the dramatic substrate-induced conformational changes involving three catalytic loops [3]. It appears that the complete active center of HPPK is assembled only after both substrates bind to the enzyme. Comparative structural analysis suggests that the structures reported here may represent an intermediate conformation required for both substrate binding and product release in the catalytic cycle
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