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Analysis of the HindIII-catalyzed reaction by time-resolved crystallography.

Kawamura T, Kobayashi T, Watanabe N - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites.DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion.In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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Affiliation: Synchrotron Radiation Research Center, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.

ABSTRACT
In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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Coordination at the metal ion-binding sites in the (a) 0 s, (b) 60 s and (c) 230 s structures. The coordinate bonds are shown as dashed lines with averaged distances (Å) of the four HindIII molecules in the asymmetric unit. The coordinate bonds from the main-chain O atom of Ala109 and from the phosphate-moiety O atom of DA5 to the Na+ ion at site A are indicated by bold lines.
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fig6: Coordination at the metal ion-binding sites in the (a) 0 s, (b) 60 s and (c) 230 s structures. The coordinate bonds are shown as dashed lines with averaged distances (Å) of the four HindIII molecules in the asymmetric unit. The coordinate bonds from the main-chain O atom of Ala109 and from the phosphate-moiety O atom of DA5 to the Na+ ion at site A are indicated by bold lines.

Mentions: In this time-resolved study, electron-density peaks at site A increase at a faster rate than at site B. At 25 s, the refined occupancies of manganese ion at site A have already become almost 1.00; the average for the four subunits is 0.98. Meanwhile, the averaged occupancies of site B in the asymmetric unit are 0.47, 0.58, 0.74 and 0.96 at 25, 40, 60 and 230 s, respectively, if the sites were refined as manganese ions. However, the behaviour of these metal sites should be reflected by the increase in manganese ion and the decrease in sodium ion over the soaking time. Since simultaneous refinement of the occupancies and B factors of the superimposed ions did not converge, the occupancies of the two metal ions were estimated when refined as manganese. If the sites are occupied by decreasing sodium and increasing manganese ions, the estimated occupancies of manganese ion are 0.06, 0.26, 0.54 and 0.93, while those of sodium ion are 0.94, 0.74, 0.46 and 0.07 at 25, 40, 60 and 230 s, respectively. As shown in Fig. 6 ▶, manganese ion replaces sodium ion and interacts with surrounding side chains at site A without inducing significant conformational changes. The coordinations of the metal ion-binding sites are conserved during the catalytic reaction. This suggests that the difference in the rate of occupation by manganese at both sites can be considered as the difference in the intrinsic association and dissociation rate constants of manganese ions and sodium ion between sites A and B, rather than a sequential binding of manganese ion, such as binding at site A inducing the binding of the second ion at site B. A possible reason for the difference in the rate constants may result from the difference in the coordination properties at the two sites. At 0 s, the mean distance between the metal and either of the O atoms in the main-chain carbonyl of Ala109 or the phosphate moiety of DA5 is approximately 2.2 Å for the structures in the asymmetric unit (Fig. 6 ▶a). These coordination distances are more suitable for manganese ion (2.15–2.10 Å) than for sodium ion (2.35–2.45 Å) (the typical distances reported are from Hsin et al., 2008 ▶). In the 230 s structure, the manganese ion is clearly coordinated to the two ligands, Ala109 and DA5, with distances similar to those in the 0 s structure, whereas the other coordinate bond lengths are shortened (Fig. 6 ▶c). Furthermore, the two ligands, i.e. O atoms from the protein main chain and the DNA backbone, constitute the diagonal of the octahedral coordination at site A. Therefore, it is likely that the faster increase in manganese ion at site A is derived from the favourable coordination distances to manganese ion than to sodium ion compared with that in site B.


Analysis of the HindIII-catalyzed reaction by time-resolved crystallography.

Kawamura T, Kobayashi T, Watanabe N - Acta Crystallogr. D Biol. Crystallogr. (2015)

Coordination at the metal ion-binding sites in the (a) 0 s, (b) 60 s and (c) 230 s structures. The coordinate bonds are shown as dashed lines with averaged distances (Å) of the four HindIII molecules in the asymmetric unit. The coordinate bonds from the main-chain O atom of Ala109 and from the phosphate-moiety O atom of DA5 to the Na+ ion at site A are indicated by bold lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321485&req=5

fig6: Coordination at the metal ion-binding sites in the (a) 0 s, (b) 60 s and (c) 230 s structures. The coordinate bonds are shown as dashed lines with averaged distances (Å) of the four HindIII molecules in the asymmetric unit. The coordinate bonds from the main-chain O atom of Ala109 and from the phosphate-moiety O atom of DA5 to the Na+ ion at site A are indicated by bold lines.
Mentions: In this time-resolved study, electron-density peaks at site A increase at a faster rate than at site B. At 25 s, the refined occupancies of manganese ion at site A have already become almost 1.00; the average for the four subunits is 0.98. Meanwhile, the averaged occupancies of site B in the asymmetric unit are 0.47, 0.58, 0.74 and 0.96 at 25, 40, 60 and 230 s, respectively, if the sites were refined as manganese ions. However, the behaviour of these metal sites should be reflected by the increase in manganese ion and the decrease in sodium ion over the soaking time. Since simultaneous refinement of the occupancies and B factors of the superimposed ions did not converge, the occupancies of the two metal ions were estimated when refined as manganese. If the sites are occupied by decreasing sodium and increasing manganese ions, the estimated occupancies of manganese ion are 0.06, 0.26, 0.54 and 0.93, while those of sodium ion are 0.94, 0.74, 0.46 and 0.07 at 25, 40, 60 and 230 s, respectively. As shown in Fig. 6 ▶, manganese ion replaces sodium ion and interacts with surrounding side chains at site A without inducing significant conformational changes. The coordinations of the metal ion-binding sites are conserved during the catalytic reaction. This suggests that the difference in the rate of occupation by manganese at both sites can be considered as the difference in the intrinsic association and dissociation rate constants of manganese ions and sodium ion between sites A and B, rather than a sequential binding of manganese ion, such as binding at site A inducing the binding of the second ion at site B. A possible reason for the difference in the rate constants may result from the difference in the coordination properties at the two sites. At 0 s, the mean distance between the metal and either of the O atoms in the main-chain carbonyl of Ala109 or the phosphate moiety of DA5 is approximately 2.2 Å for the structures in the asymmetric unit (Fig. 6 ▶a). These coordination distances are more suitable for manganese ion (2.15–2.10 Å) than for sodium ion (2.35–2.45 Å) (the typical distances reported are from Hsin et al., 2008 ▶). In the 230 s structure, the manganese ion is clearly coordinated to the two ligands, Ala109 and DA5, with distances similar to those in the 0 s structure, whereas the other coordinate bond lengths are shortened (Fig. 6 ▶c). Furthermore, the two ligands, i.e. O atoms from the protein main chain and the DNA backbone, constitute the diagonal of the octahedral coordination at site A. Therefore, it is likely that the faster increase in manganese ion at site A is derived from the favourable coordination distances to manganese ion than to sodium ion compared with that in site B.

Bottom Line: The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites.DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion.In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Synchrotron Radiation Research Center, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.

ABSTRACT
In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Show MeSH