More Cations Bound by Avian Sarcoma Virus Integrase

 Home Page National Cancer Institute Macromolecular Crystallography Laboratory Retroviral Integrase Project NCI-Frederick MCL - Protein Structure Section
Structures determined by the Macromolecular Crystallography Laboratory, Protein Structure Section at the NCI-Frederick campus. Work performed in collaboration with the Skalka Laboratory in the Institute for Cancer Research, Fox Chase Cancer Center.




ASV active site with different cations


This view of the active site shows the "apoenzyme" (only protein in water) structure in the lower righthand corner, with water molecules shown as small spheres. The three active site residues (D64, D121, and E157) are shown along with the N153 residue at bottom center and a water molecule, which help to stabilize this part of the protein. The essential metal cations believed to activate the enzyme in vivo (in the cell) are manganese (Mn, large purple sphere) or magnesium (Mg, large blue sphere), each with four close coordinating water molecules (smaller spheres). Another structure was solved with two zinc cations coordinated in the active site, shown here as yellow spheres. The conditions were similar, as all crystals were soaked in solutions containing 100 millimolar concentrations of salts. (Significantly higher concentrations of MnCl2 or MgCl2 salts were also tried, up to 500 millimolar; only one metal cation was ever seen bound to the protein using these salts.)

There is a controversy about the number of metal ions required to perform the biochemical reaction (DNA integration). Some reports suggest that one is enough; others suggest that two metal ions are required. These discussions have not been resolved, even though we have observed two metals bound to IN. Magnesium or manganese (separately) each are adequate to allow full activity, even though we have observed only one metal bound to the active site. Although we see two zinc ions bound to the active site, this metal carries out only the first of the integration reactions. See also the details of the integration reaction mechanism.





The electron density map around the active site is shown in brown for Mg or Mn, and the metal ions are shown as blue spheres. Either will activate IN, although it is presumed that magnesium is the activating cation in the cells, since it is present at high concentrations.






The electron density map is shown in blue for zinc ions (yellow spheres). Zinc also activates the first of two IN reactions.








The electron density map is shown in green for cadmium ions (purple spheres). Cadmium does not activate IN, even though it is a divalent cation like zinc and magnesium.






Calcium (brown sphere) binds to ASV IN but does not allow enzymatic activity, like cadmium and unlike zinc.
It is not known why certain metal cations act as cofactors for catalytic activity and other types of metal cations do not. Some possible reasons include size, strength of the electric field created by the ion, or the number of water molecules coordinated around the metal ions. (One water molecule is "activated" by IN and reacts directly with viral DNA in the "processing" part of the integration reaction. Viral DNA is then activated to insert into host cell DNA in the "joining" reaction.)


All of the protein atoms are shown in the same colors (carbon, green; oxygen, red; water molecules, red spheres). One water molecule could not be assigned to either of the two zinc cations but was present between them in the electron density; the average position is marked with a pink sphere.


Click here for more information about the interaction between the essential cations and ASV IN.

The coordinate and image files of ASV IN with these bound metal cations are available from the PDB.

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If you have questions or comments about the Integrase Project web site,
contact: Jerry N. Alexandratos at alexandr@ncifcrf.gov.