Binding of Different Divalent Cations to the Active Site of Avian Sarcoma Virus Integrase and Their Effects on Enzymatic Activity

Abstract

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called “processing,” and the second is a polynucleotide transfer or “joining” step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences. Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called “processing,” and the second is a polynucleotide transfer or “joining” step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences. The retroviral integrase (IN) 1The abbreviations used are: IN, integrase; HIV, human immunodeficiency virus; ASV, avian sarcoma virus. is a virus-encoded enzyme that catalyzes integration of viral DNA into host DNA (1Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (537) Google Scholar, 2Goff S.P. Annu. Rev. Genet. 1992; 26: 527-544Crossref PubMed Scopus (218) Google Scholar, 3Vink C. Groeneger O.A.M. Plasterk R.H. Nucleic Acids Res. 1993; 21: 1419-1425Crossref PubMed Scopus (250) Google Scholar). As DNA integration is an essential step in the virus life cycle, IN is an important target for the design of drugs that block the replication of pathogenic retroviruses such as the human immunodeficiency virus (HIV). The integration reaction occurs in two distinct steps. First, IN nicks the viral DNA near the 3′-ends of both strands (the “processing” reaction); it then inserts these ends into host target DNA (the joining reaction). Both reactions comprise a nucleophilic attack by a hydroxyl oxygen on a phosphorous atom in the DNA backbone; the hydroxyl is derived from a water molecule in the processing reaction and from the newly formed 3′-OH at the end of the viral DNA in the joining reaction. Divalent cations Mn2+ or Mg2+ are known to be required as cofactors. In vitro the reactions are most efficient in the presence of Mn2+, but as Mg2+ is more abundant in living cells, it is generally presumed to be the physiologically relevant cation. Retroviral integrases contain approximately 300 amino acids and are composed of three domains (4Andrake M.D. Skalka A.M. J. Biol. Chem. 1996; 271: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The first two domains are highly conserved, and both include metal-binding sites. The N-terminal domain (amino acids ∼1–50) contains a zinc finger-like motif, HHCC. Binding of Zn2+ at this site stabilizes the structure of HIV-1 IN and enhances multimerization and activity (5Lee S.P. Xiao J. Knutson J.R. Lewis M.S. Han M.K. Biochemistry. 1997; 36: 173-180Crossref PubMed Scopus (158) Google Scholar, 6Lee S.P. Han M.K. Biochemistry. 1996; 35: 3837-3844Crossref PubMed Scopus (68) Google Scholar, 7Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13639-13664Google Scholar). The central, catalytic domain (amino acids ∼50–200) is characterized by a triad of invariant acidic amino acids (Asp-64, Asp-121, and Glu-157 in ASV IN), the last two separated by 35 amino acids comprising the D,D(35)E motif. These three acidic residues are essential for both processing and joining activity and have been proposed to bind the divalent metal cofactors during catalysis (8Kulkosky J. Jones K.S. Katz R.A. Mack J.P. Skalka A.M. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (524) Google Scholar). Solution of the crystal structures of the isolated catalytic core domains of HIV-1 (9Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (730) Google Scholar, 10Bujacz G. Alexandratos J. Zhou-Liu Q. Clement-Mella C. Wlodawer A. FEBS Lett. 1996; 398: 175-178Crossref PubMed Scopus (105) Google Scholar) and ASV IN (11Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (201) Google Scholar, 12Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) have revealed that these retroviral enzymes belong to a superfamily of nucleases and polynucleotidyltransferases, all of which contain a cluster of conserved acidic amino acids at their presumed active sites. HIV-1 reverse transcriptase ribonuclease H (RNase H) domain, another member of this superfamily, was shown to bind two divalent cations in this site (13Davies J.F., II Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (536) Google Scholar), prompting the suggestion (14Yang W. Steitz T.A. Structure ( Lond. ). 1995; 3: 131-134Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) that all members of this family may use a two-metal catalytic mechanism like that deduced for the 3′-5′ exonuclease of Escherichia coli DNA polymerase I (15Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (943) Google Scholar, 16Rice P. Craigie R. Davies D.R. Curr. Opin. Struct. Biol. 1996; 6: 78-83Crossref Scopus (187) Google Scholar). We have shown that side chains of two of the acidic triad residues in the D,D(35)E motif in ASV IN also form a metal binding pocket. A single ion, Mn2+ or Mg2+, is complexed to Asp-64 and Asp-121 when crystals of the isolated catalytic domain of ASV IN are soaked in metal-containing solutions (12Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We hypothesized that a second metal be bound the Asp-64 and Glu-157 in the full-length or in the presence of we show that divalent cations can also bind in the active site of crystals of the isolated catalytic core domain of ASV IN. in the of Zn2+ and two are complexed by side chains from all three of the acidic amino acids of the D,D(35)E motif. the of these we enzymatic in the presence of these with the isolated catalytic full-length ASV IN and an N-terminal derivative that the zinc finger motif but both processing and joining The results of the are consistent with a two-metal reaction The show that whereas Zn2+ is a cofactor for the it or for the activities of IN. and of the catalytic core domain of ASV IN have been (11Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (201) Google Scholar), and the crystals used by the same of divalent cations used in this in the to the The crystals soaked for as in the previous (12Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). on crystals in the presence of divalent cations in their of binding at on or with and with Z. for Scholar). with > used in and the was either or Structure the PubMed Scopus Google Scholar), which was to structures with of the and for that whereas of the Mn2+ and Mg2+ whereas structures in the of the metal-binding with to is in for these which are to in the results The of the with Ca2+, Zn2+, and Cd2+ have been with the and The of Mn2+ (12Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google for ASV IN complexed with divalent I II soaked in metal at the of the is for bind to the residues in to as are in the of are in are to be for the of in a soaked in metal at the of the is for bind to the residues in to as are in the of are in are to be for the of The full-length ASV IN and the catalytic as R.A. Merkel G. Skalka A.M. 1996; PubMed Scopus Google Scholar). as (11Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (201) Google Scholar). for and the was an to the end of ASV The of the was with The reaction and in processing and the of IN was and the DNA was the of IN was and the DNA was The was by DNA strands of and with the with at the and metal on and a are as of of IN or IN In we soaked crystals of the isolated ASV IN catalytic domain with the two known divalent cation Mn2+ and G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We of both cations Asp-64 and Asp-121 of the catalytic triad but of the of Glu-157 in metal we a a of divalent cations and a of of the catalytic domain of ASV soaked in solutions of divalent Mg2+, Mn2+, Zn2+, Ca2+, and of the crystals and at As the to the structure of the Mg2+ at the of is and a cation bound the of Asp-64 and Asp-121 of the D,D(35)E motif site However, of binding of a second cation be in this was at a of Mg2+ that binding was A cluster However, a single metal was at the crystals in an of metal-binding in A for Mn2+ is consistent with that ASV IN is approximately more active in Mn2+ soaked in also to bind a divalent cation at site with the and the active site residues similar to with Mg2+ and Ca2+, is an in a with the metal in the of the and three water in the we that Zn2+ in on the of the isolated catalytic domain of ASV IN with at a as as and of the at the of Zn2+, two of the bind in the catalytic at site I Asp-64 and Asp-121 and a site coordinated by Asp-64 and Glu-157 The the zinc cations in the active site is is in a by amino acids with the coordinated to coordinated to a water and with two water The zinc is bound in the of the of the catalytic domain and is coordinated by residues and bound at and show whereas the cations in I and II are coordinated to two of the and water molecule water molecule may a of cations in the catalytic by both zinc water is clearly in the it as a in the the water molecule in this from of Zn2+ to the it was in the with to As this water molecule it is that is The water the zinc are in the crystal soaked in Zn2+, we a of metal binding similar to that at In this both catalytic binding showed with site II site are for site II for site However, the structure with Zn2+ and for both cations in the active Cd2+ in the catalytic in and coordinated to all three residues of the essential triad However, was of site II and for this for the in site is similar to binding at the of Zn2+ and that the cations in site II may be bound site The the cadmium in the catalytic is The Cd2+ in site I has an similar to the of Mn2+ and The Cd2+ in site II has a water with the first cation. in this both Glu-157 with this cation. The most water to bound to the Asp-64 have and The of the three acids are when or more cations are is in the of the side of Asp-64 of the from their for all structures in is a of the side of Asp-121 and a more in of the side of Glu-157 The of the side of Glu-157 results in approximately the same in as in two of ASV IN from and (11Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (201) Google Scholar). The in metal binding is consistent with the that single in these residues activity of both ASV and HIV-1 IN (8Kulkosky J. Jones K.S. Katz R.A. Mack J.P. Skalka A.M. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (524) Google Scholar). The Zn2+ structures show the active that are with two metal cations at which cations are by the we soaked ASV IN catalytic domain crystals in of Zn2+ either Mg2+ or In the for both structures to the structure with Zn2+ However, a Zn2+, Mn2+ structure at of a The water the metal active site I as a of both Zn2+ and Mn2+ We these as a of Zn2+ and Mn2+ in site I and Zn2+ in site As in the of zinc cations in I and II are to be may the of the such that site II is by Zn2+, then site I can be by Mn2+ or Ca2+, Zn2+, and Cd2+ of site I I and in the active site of the catalytic domain, we of these cations as cofactors for enzymatic activity of IN. The isolated catalytic core domain of ASV IN two a DNA and a DNA activity (11Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (201) Google Scholar, J. Katz R.A. Merkel G. Skalka A.M. 1995; PubMed Scopus Google Scholar). DNA by the ASV IN catalytic domain is efficient and similar in specific activity to that of the full-length we first for this which is characterized by cleavage the and A of the conserved near the viral DNA endonucleolytic activity is distinct from and is The catalytic core domain was with a DNA that a viral DNA end in the presence of of and as as the known metal and the Zn2+ activity As shown in the activity a at approximately similar results with this activity is approximately of that with the same of However, activity is at The of is approximately with in activity to The activity at is to the as similar results with the and We that the ASV IN catalytic domain activity with Zn2+, as a cofactor at a in which site I is to be and site II is to be at of Zn2+ are Mg2+ and bind to site I and Cd2+ bind to I and II in the activity be in the presence of a of of these We the activity of the catalytic domain in the presence of Zn2+, Mg2+, and The metal used and As in activity is in the presence of although the and of the reaction are approximately that with However, activity was with Thus, at for the isolated catalytic core domain, we activity with the cation presumed to be important in but activity with Zn2+ as a The ASV catalytic domain was also for activity which at of the with the full-length In the presence of is clearly with the catalytic domain, as J. Katz R.A. Merkel G. Skalka A.M. 1995; PubMed Scopus Google Scholar). However, activity was with of the We that Zn2+ is to activity of the isolated catalytic domain these As of Zn2+ and Mn2+, we of site I by Mn2+ and of site II by Zn2+. activity a but of Mn2+ in the presence of of the divalent to be and of the was as in and We a in activity at or of Mg2+ to Mn2+ As Mg2+ activity on the of this is at the same and was a in activity at of Mg2+ or In to results with Mg2+ and Ca2+, both and showed of activity of Zn2+ and Mn2+, the reaction was approximately The activity at this may the are these results are consistent with of the activity in of the activity. by Zn2+ is also consistent with the of the isolated catalytic domain for Zn2+ as by in the crystals at metal which can also bind to I and II but is a cofactor for by the isolated catalytic core domain, is a more activity was with this divalent cation at the we that the the of these to bind to the catalytic these results a to Zn2+ > Ca2+, Mg2+ and is consistent with the results shown in We the divalent as a cofactor for the processing and joining activities of full-length ASV IN. Our revealed activity with full-length IN in a of of or Cd2+ Zn2+ activity with an of We then the activities in the presence of or (the As in the full-length IN showed processing activity in the presence of this activity is with that with Mg2+, but both are approximately that with Mn2+ as the cofactor activity can be as into the viral DNA which a of that is the As joining activity is in the presence of joining activity was with Zn2+. We showed that a ASV IN derivative lacking the N-terminal zinc binding domain, near of processing and joining activity in the presence of R.A. Merkel G. Skalka A.M. 1996; PubMed Scopus Google Scholar). As shown in and the processing and joining activities of in also similar to that of the full-length IN. processing activity can also be with in the presence of Zn2+ these we that the N-terminal domain is required for the processing activity. However, by of A and of that of the N-terminal domain in in the presence of Zn2+. two for Zn2+ in these a in the N-terminal domain, as is required for and a catalytic as the cation We also the for activity. As shown in this activity was in Mn2+, as shown in Mg2+ was whereas activity was in of the by that the of in Zn2+ was by approximately with Mn2+ and with these we that activity of as with the full-length protein, Zn2+ also to joining activity by enzymes active in the have an for divalent However, of the of such cations have been in a of the In the of ASV IN, we binding of Mn2+ and Mg2+ to a single site Asp-64 and Asp-121 we report the binding of two zinc and two cadmium to I and II are Asp-64 and a for this member of the invariant triad in the D,D(35)E motif. more are from the active site of the As it is known these can be in the full-length protein, the of this binding be is that metal bound to or both of these a in activity as for enzymes C. M.D. S.P. W. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). is the with divalent cations show of site of the two contains two to the but in all structures of the isolated catalytic domain, the for Glu-157 are for the two in the active site, of that side may more for binding of the coordinated Mg2+, Mn2+, or the coordinated Zn2+, which has for the of binding site and a Katz J.P. J. Chem. 1995; Scopus Google Scholar). the coordinated Cd2+ bind in site but we that this is to both of Glu-157 for and this The of zinc in the active site with the of in or Mg2+ by and Biochemistry. 1993; PubMed Scopus Google Scholar). As is the both are to (the is in the and they are by the of an have been in the for such as S. A. C. Z. PubMed Scopus Google Scholar) and A. A. F. EMBO J. 1991; 10: PubMed Scopus Google Scholar). In with these is also of a water molecule the two although this water is in ASV IN. In the of a bound DNA the to specific metal binding to site II and the mechanism by which or transfer occurs with ASV IN. and Steitz (14Yang W. Steitz T.A. Structure ( Lond. ). 1995; 3: 131-134Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) have the of the cluster of acidic residues the active of enzymes in the superfamily to which integrase and more of metal of of the members of this family by (12Bujacz G. Jaskólski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure ( Lond. ). 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The binding of two metal in the ASV IN active site with the 3′-5′ exonuclease domain of the coli DNA polymerase I in which two are proposed to to an and a DNA in the active site (14Yang W. Steitz T.A. Structure ( Lond. ). 1995; 3: 131-134Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (943) Google Scholar). In we metal of the active of ASV IN, HIV-1 reverse transcriptase and the exonuclease domain of the of DNA polymerase I (15Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (943) Google Scholar). in of the three the in of both divalent cations and the is In this site I of ASV IN is on site A of the both of which have been shown to have similar metal binding in of and metal of site I of ASV IN with site of the exonuclease domain as a of the these enzymes are also a of divalent cations is site A in the is by a Zn2+ ion, whereas site is by Mg2+, with in the coordination. is the for Zn2+ clearly is in both metal both with coordination. As are two binding ion, are that or of coordination. These may be to the of by of ASV IN or the DNA both integrase and the of Zn2+ to binding is more an of that for that contain more of divalent cation are in enzymes that The second metal is Mg2+ for in or S. A. C. Z. PubMed Scopus Google Scholar, A. A. F. EMBO J. 1991; 10: PubMed Scopus Google Scholar). is important to that active site from shown in are for enzymes with or activity. divalent cations are in of the structures of polymerase J.F., II Hostomska Z. R.A. Hostomsky Z. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), separated by and also coordinated by three acidic we on the active site of more is the active site of H Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) the two Mg2+ are with of coordinated by a single oxygen and whereas the is by water and with These show the of the and are a that be Our show that although Zn2+ is Mn2+, it can serve as a cofactor for activity of the isolated catalytic core domain of ASV IN, whereas is activity with Mg2+, Ca2+, or of activities in of Mn2+ and the cations that both Zn2+ and Cd2+ bind with Mn2+ and that Mg2+ and bind with is consistent with the of these in of the catalytic domain. We also a for the of Zn2+ at The for this activity at Zn2+ is However, the most from these was the that Zn2+ the activity of the catalytic domain, whereas Mg2+, the presumed physiologically relevant to Zn2+ or of the catalytic domain binding cations serve as cofactors for processing and joining by the full-length As ASV IN can both processing and joining in in the of the N-terminal domain it is to the presumed of Zn2+ bound to the N-terminal domain from a catalytic We that both full-length ASV IN and which the N-terminal motif, both and endonucleolytic processing activity. The activity is with the full-length with that the N-terminal domain is required for activity. both full-length and showed processing activity to that with have that the of Zn2+ can the activity of HIV-1 IN S.P. Han M.K. Biochemistry. 1996; 35: 3837-3844Crossref PubMed Scopus (68) Google Scholar, 7Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13639-13664Google Scholar). However, in their was shown to be by the N-terminal zinc binding domain. Thus, is the first report that Zn2+ can also as a cofactor for catalysis by a retroviral In to results with we joining activity by either full-length ASV IN or in the presence of Zn2+. We also that Zn2+ to activity by either the catalytic core domain or Thus, the two reactions in which the is derived from a DNA and with DNA is required joining and are impaired with Zn2+ as a As the same triad of acidic amino acids in the catalytic is essential for both processing and joining in the presence of Mn2+ and Mg2+ and these steps employ similar is for this is that the of Zn2+ with Mn2+ or Mg2+ with target or to the that target DNA to the for these distinct metal preferences. the that to the active site but can step in integration may be relevant to the design of active site of retroviral integrases. We the of M. Jaskólski in the of this We and R. for the of and G. for the for of the