(Received for publication, March 4, 1997, and in revised form, April 22, 1997)
From the Macromolecular Structure Laboratory,
NCI-Frederick Cancer Research and Development Center, ABL-Basic
Research Program, Frederick, Maryland 21702, and the
§ Faculty of Food Chemistry and Biotechnology, Technical
University of
ód
,
ód
, Poland
From the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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)1 is a
virus-encoded enzyme that catalyzes integration of viral DNA into host
DNA (1-3). 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 (4). 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 (5-7). 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 (8).
Solution of the crystal structures of the isolated catalytic core
domains of HIV-1 (9, 10) and ASV IN (11, 12) 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 (13), prompting the suggestion (14) 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
(15, 16). 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 (12). We
hypothesized that a second metal might be bound between the Asp-64 and
Glu-157 in the full-length protein or in the presence of substrate.
Here we show that additional divalent cations can also bind in the active site of crystals of the isolated catalytic core domain of ASV IN. Moreover, in the case of Zn2+ and Cd2+, two ions are complexed by side chains from all three of the acidic amino acids of the D,D(35)E motif. To investigate the significance of these observations, we measured enzymatic properties in the presence of these metals with the isolated catalytic core, full-length ASV IN protein and an N-terminal deletion derivative that lacks the zinc finger motif but retains both processing and joining activities. The results of the structural analysis are consistent with a two-metal reaction mechanism. The biochemical studies show that whereas Zn2+ is a cofactor for the hydrolytic, nicking activities, it provides little or no detectable support for the polynucleotidyltransferase activities of IN.
Expression, purification, and crystallization of the catalytic core domain of ASV IN have been described previously (11), and the crystals used here were grown by the same procedures. Salts of divalent cations used in this study were dissolved in the well buffer to produce the soaking solutions. The crystals were soaked for 2-3 days, as in the previous studies (12). Some experiments were performed on crystals grown directly in the presence of divalent cations without finding any difference in their mode of binding (data not shown).
Diffraction data were collected at room temperature on MAR300 or
DIP2020 image plate detectors using graphite-monochromated CuK radiation. They were processed with DENZO and scaled with SCALEPACK (17). Data with F > 2
(F)
were used in refinement, and the low resolution cutoff was either 8 or
10 Å. Structure refinements were carried out using the program PROLSQ
(18), which was modified to utilize free R-factor
computation. Some structures were refined with 8% of the separately
and randomly chosen data reserved for that purpose, whereas other
refinements did not utilize free R-factor (see Table I).
Structures of the Mn2+ and Mg2+ complexes were
refined very carefully until final convergence, whereas other
structures were modeled carefully only in the vicinity of the
metal-binding sites, with other areas subject to less careful rebuilding. This is reflected in somewhat higher R-factors
for these structures, which are nevertheless adequate to impart
confidence in the structural results described here. The coordinates of
the complexes with Ca2+, Zn2+, and
Cd2+ have been deposited with the Protein Data Bank,
Brookhaven National Laboratory, Upton, NY (accession codes 1VSI, 1VSH,
and 1VSJ, respectively). The coordinates of selected Mg2+
and Mn2+ complexes were deposited previously (12).
|
The
full-length ASV IN and the catalytic IN-(39-286) were purified as
described previously (19). Assays were carried out exactly as described
(11). Briefly, for nicking and processing, the substrate was an 18-base
pair duplex corresponding to the U3 end of ASV DNA. The 5-end of the
plus strand was labeled with 32P. The reaction mix
contained 50 mM Tris, pH 8.4, 2 mM
-mercaptoethanol, 200 µg/ml bovine serum albumin, 50 mM NaCl, and 4% glycerol. Reactions were carried out in
substrate excess. For processing and joining, the concentration of IN
was 2 µM, and the DNA substrate was 15 µM.
For disintegration reactions, the concentration of IN was 1 µM, and the DNA substrate was 8 µM. The
disintegration substrate was prepared by annealing four separate DNA
strands of 19, 16, 10, and 9 nucleotides with the 10-nucleotide strand
end-labeled with 32P. Reactions were carried out at the
indicated times and metal concentrations. Reaction products were
fractionated on 20% acrylamide-urea sequencing gels, and bands were
quantified using a Fuji phosphoimager. Results are expressed as µmol
of product/µmol of IN monomer or IN fragment.
In our earlier studies, we soaked crystals of the isolated ASV IN catalytic domain with the two known divalent cation cofactors, Mn2+ and Mg2+ (12). We observed coordination of both cations between Asp-64 and Asp-121 of the catalytic triad but no participation of its third member, Glu-157. To further investigate the possible participation of Glu-157 in metal binding, we carried out a systematic analysis encompassing a variety of divalent cations and a wider range of concentrations.
Crystals of the catalytic domain of ASV IN-(52-207) were soaked in
2-500 mM solutions of five divalent cations:
Mg2+, Mn2+, Zn2+, Ca2+,
and Cd2+. Structures of the metal-soaked crystals were
solved and refined at moderately high resolution (Table
I). As reported earlier, the electron density map
corresponding to the structure of the Mg2+ complex obtained
at the highest concentration of its salt is exceptionally clear and
shows a cation bound between the carboxylates of Asp-64 and Asp-121 of
the D,D(35)E motif (denoted site I). However, no indication of binding
of a second cation could be found in this map. Less than 30% occupancy
was observed at a lower concentration of Mg2+ (20 mM), indicating that binding was much weaker. A putative metal/water cluster did not refine well. However, a similarly positioned, single metal ion was detected at full occupancy after soaking the crystals in an even lower (10 mM)
concentration of MnCl2. No additional metal-binding sites
were observed, even in 500 mM MnCl2 (Fig.
1A). A higher apparent affinity for
Mn2+ is consistent with observations that ASV IN is
approximately 30-fold more active in Mn2+ than
Mg2+.
Crystals soaked in 100 mM CaCl2 were also found to bind a divalent cation at site I, with interactions between the calcium ion and the active site residues similar to those observed with Mg2+ and Mn2+. With Ca2+, coordination is an incomplete octahedron in appearance, a square pyramid with the metal in the center of the square base and three water molecules in the coordination sphere.
Zn2+ and Cd2+ Bind at Multiple Sites in the ASV IN Catalytic DomainUnexpectedly, we found that Zn2+ binds in four separate sites on the surface of the isolated catalytic domain of ASV IN (Table I), with partial occupancy observed at a concentration as low as 2 mM and full occupancy of the sites at 100 mM. At the higher concentration of Zn2+, two of the ions bind in the catalytic center, one at site I between Asp-64 and Asp-121 and a second, denoted site II, coordinated by Asp-64 and Glu-157 (Fig. 1B). The distance between the zinc cations in the active site is 3.62 Å. Site III (not shown) is in a loop defined by amino acids 92-107, with the ion directly coordinated to His-103, coordinated to His-93 via a water molecule, and liganded with two other water molecules. The fourth zinc ion is bound in the vicinity of the C terminus of the catalytic domain and is coordinated by residues His-198 and Tyr-194 (site IV).
Zinc ions bound at sites III and IV show clear tetrahedral
coordination, whereas the cations in sites I and II are coordinated to
two oxygens of the carboxylates and one water molecule each. One
additional water molecule may complete a tetrahedral coordination of
cations in the catalytic center by bridging both zinc ions. This
putative water is not seen clearly in the electron density maps; it
does, however, appear as a weak peak in 2Fo F1ac maps. During the refinement process, the water molecule located in this position shifts from one edge of one Zn2+ electron density to the other, so it was placed in the
final model with null occupancy just to indicate its putative
placement. As this water molecule faces bulk solvent, it is not
surprising that its position is not well defined. The other water
molecules coordinating the zinc ions are found in very clear electron
density. With the crystal soaked in 2 mM Zn2+,
we observed a pattern of metal binding similar to that at 100 mM. In this case, both catalytic binding sites showed less
than complete occupancies, with site II lower than site I. Temperature factors are correspondingly higher for site II than for site I. However, the structure with 100 mM Zn2+ had
complete occupancy and relatively low temperature factors for both
cations located in the active site.
Two Cd2+ ions were visible in the catalytic center after
soaking in 100 mM CdCl2 and were again
coordinated to all three carboxylate residues of the essential triad
(Fig. 1C). However, there was lower occupancy of site II and
higher temperature factor for this ion than for the ion in site I. This
is similar to binding observed at the lowest concentration (2 mM) of Zn2+ and again suggests that the cations
in site II may be bound less strongly than those occupying site I. The
distance between the cadmium ions in the catalytic center is 4.06 Å (see Fig. 2). The Cd2+ in site I has an
almost perfect octahedral coordination sphere, very similar to the
coordination of Mn2+ and Mg2+. The
Cd2+ in site II has deformed octahedral coordination,
sharing a water ligand with the first cation. Singularly in this case,
both Glu-157 carboxyl oxygens coordinate with this one cation. The most
solvent-accessible water molecules liganded to each metal, bound
opposite to the Asp-64 ligand, have slightly longer hydrogen bonding
distances and higher temperature factors.
The atomic coordinates of the three crucial carboxylic acids are only slightly affected when one or more cations are bound. There is practically no change in the position of the side chain of Asp-64 (mean shift of the atomic positions from their average values, calculated for all structures listed in Table I, is 0.125 Å), a very slight movement of the side chain of Asp-121 (0.187 Å), and a more pronounced difference in location of the side chain of Glu-157 (0.857-Å shift) (Fig. 2). The rotation of the side chain of Glu-157 results in approximately the same difference in carboxylate positions as seen in two different, uncomplexed forms of ASV IN crystallized from different precipitants, polyethylene glycol and ammonium sulfate (11). The minimal deviation in carboxylate positioning upon metal binding is consistent with the observation that even single conservative substitutions in these residues drastically reduced activity of both ASV and HIV-1 IN (8).
Competition for Cation Binding at the Active SiteThe Zn2+ structures show the only active sites that are fully occupied with two metal cations at 100 mM concentration (Table I). To determine which cations are preferred by the enzyme, we soaked ASV IN catalytic domain crystals in various concentrations of Zn2+ plus either Mg2+ or Mn2+ salts. In the highest concentration (100 mM for each salt), both structures were essentially identical to the structure with 100 mM Zn2+ alone. However, a Zn2+, Mn2+ structure at 10 mM concentrations of each salt produced a mixed image. The water coordination around the metal occupying active site I appears as a superposition of both Zn2+ and Mn2+ structures. We interpreted these maps as a mixture of Zn2+ and Mn2+ in site I and only Zn2+ in site II. As summarized in Table I, the occupancy of zinc cations in sites I and II are refined to be equal. This result may indicate the existence of some cooperativity between the sites, such that if site II is occupied by Zn2+, then site I can no longer be occupied by Mn2+ or Mg2+.
Activity of the ASV IN Catalytic Domain with Various Divalent CationsHaving observed Ca2+,
Zn2+, and Cd2+ occupancy of site I (or I and
II) in the active site of the catalytic domain, we asked whether any of
these cations could function as cofactors for enzymatic activity of IN.
The isolated catalytic core domain of ASV IN displays two
metal-dependent activities, a DNA endonuclease
"nicking" activity, and a DNA cleavage-ligation
"disintegration" activity (11, 20). DNA nicking by the ASV IN
catalytic domain is quite efficient and similar in specific activity to
that of the full-length protein. Therefore, we first assayed for this
nicking activity, which is characterized by preferred cleavage between
the C and A of the conserved CA dinucleotide near the viral DNA termini
(Fig. 3A, 3). This
endonucleolytic activity is distinct from "
2" processing, and its
biological relevance is not yet understood. The catalytic core domain
was incubated with a short DNA duplex substrate that represents a viral
DNA end in the presence of varying concentrations of ZnCl2,
ZnSO4, CaCl2, and CdSO4 as well as
the known metal cofactors, MgCl2 and MnCl2. Of
the new metals tested, only Zn2+ supported significant
activity (comparison not shown). As shown in Fig. 3A, the
Zn2+-dependent activity exhibits a sharp peak
at approximately 2 mM ZnSO4; similar results
were obtained with ZnCl2 (data not shown). At this peak,
activity is approximately half of that observed with the same
concentration of MnCl2. However, significantly less activity is observed at higher ZnSO4 concentrations. The
optimum concentration of MnCl2 is approximately 10 mM, with little change in activity up to 25 mM.
The decreased activity at ZnSO4 concentrations >2
mM is not due to the anion, as similar results were
observed with the chloride and sulfate salts. We conclude that the ASV IN catalytic domain displays significant nicking activity with Zn2+, as a cofactor at a concentration in which site I is
likely to be fully occupied and site II is likely to be at least
partially occupied. Higher concentrations of Zn2+ are
inhibitory. Although Mg2+ and Ca2+ could bind
to site I and Cd2+ could bind to sites I and II in the
crystal, no nicking activity could be detected in the presence of a
broad range of concentrations of these metals (data not shown).
We compared the time-dependent nicking activity of the catalytic domain in the presence of Zn2+, Mg2+, and Mn2+. The metal concentrations used were 2 mM ZnSO4, 10 mM MnCl2 (the optimal concentrations determined above), and 5 mM MgCl2. As illustrated in Fig. 3B, nicking activity is readily detected in the presence of ZnSO4, although the rate and extent of the reaction are approximately one-tenth that observed with Mn2+. However, no activity was detected with MgCl2. Thus, at least for the isolated catalytic core domain, we find no activity with the cation presumed to be important in vivo but significant activity with Zn2+ as a cofactor.
The ASV catalytic domain was also assayed for disintegration activity (Fig. 3C), which proceeds at 0.2% of the rate observed with the full-length protein. In the presence of 10 mM MnCl2, disintegration is clearly detectable with the catalytic domain, as described previously (20). However, no disintegration activity was observed with 2 mM ZnSO4, even after prolonged exposure of the autoradiogram. We conclude that Zn2+ is unable to support significant disintegration activity of the isolated catalytic domain under these conditions.
Effects of Metal Combinations on the Nicking Activity of the Catalytic CoreAs noted above, using equimolar (10 mM) amounts of Zn2+ and Mn2+, we
observed predominant occupancy of site I by Mn2+ and
exclusive occupancy of site II by Zn2+. For comparative
activity studies, incubations were carried out using a fixed but
suboptimal concentration of Mn2+ (3 mM) in the
presence of increasing concentrations of the divalent metals to be
tested, and production of the 3 product was followed as in Fig. 3,
A and B. We observed a slight increase in
activity at low or equal concentrations of Mg2+ relative to
Mn2+ (Fig. 4). As Mg2+ does not
support nicking activity on its own, the significance of this increase
is not yet clear. Ca2+ had no significant effect at the
same concentrations, and there was only a slight decrease in activity
at higher concentrations of Mg2+ or Ca2+.
In contrast to results with Mg2+ and Ca2+, both ZnCl2 and ZnSO4 showed potent inhibition of Mn2+-dependent nicking activity (Fig. 4). At equal concentrations of Zn2+ and Mn2+, the reaction was inhibited approximately 75%. The residual activity observed at this concentration may reflect the Zn2+-dependent nicking. Although other interpretations are possible, these results are consistent with inhibition of the Mn2+-dependent activity in favor of the Zn2+-dependent activity. This potent inhibition by Zn2+ is also consistent with the apparent high affinity of the isolated catalytic domain for Zn2+ ions, as exemplified by high occupancy in the crystals even at relatively low metal concentrations. Cd2+, which can also bind to sites I and II but is not a cofactor for nicking by the isolated catalytic core domain, is even a more potent inhibitor than Zn2+; no significant activity was detected with this divalent cation at the higher concentrations (Fig. 4). If we assume that the inhibition reflects the abilities of these metals to bind to the catalytic center, these results suggest a relative affinity corresponding to Cd2+, Zn2+ > Mn2+ > Ca2+, Mg2+ and is consistent with the results shown in Table I.
Zn2+ Can Serve as a Cofactor for the Processing Activity of Full-length ASV INWe next asked if Zn2+
or the other previously untested divalent metals could function
as a cofactor for the processing and joining activities of full-length
ASV IN. Our initial survey revealed no significant activity with
full-length IN in a range of concentrations of Ca2+ or
Cd2+ (data not shown). Zn2+ did support
activity with an optimal concentration of 2 mM (not shown).
We then compared the activities in the presence of 2 mM ZnSO4 or 10 mM MgCl2 (the optimal
concentration, data not shown). As illustrated in Fig.
5A, the full-length IN showed significant processing activity (2 nicking) in the presence of Zn2+;
this activity is slightly reduced compared with that observed with
Mg2+, but both are approximately 10-fold lower than that
observed with Mn2+ as the cofactor (not shown). Joining
activity can be detected as insertion events into the viral DNA
substrate, which produces a ladder of products that is longer than the
substrate (Fig. 5C). As expected, joining activity is
readily detected in the presence of Mg2+; however, no
significant joining activity was observed with Zn2+.
The N-terminal Domain Is Not Required for Zinc-dependent Processing Activity
We previously showed that IN-(39-286), a non-fused ASV IN derivative lacking the N-terminal zinc binding domain, displays near wild type levels of processing and joining activity in the presence of Mn2+ (19). As shown in Fig. 5 (B and D), the processing and joining activities of IN-(39-286) in Mg2+ are also similar to that of the full-length IN. Near wild type processing activity can also be detected with IN-(39-286) in the presence of Zn2+ (Fig. 5B). From these results, we conclude that the N-terminal domain is not required for the zinc-dependent processing activity. However, quantitation by phosphoimaging of panels A and B of Fig. 5 indicates that deletion of the N-terminal domain does cause some (~70%) reduction in apparent rates in the presence of Zn2+. This suggests two roles for Zn2+ in these experiments: a structural role in stabilizing the N-terminal domain, which, as our comparison indicates, is required for optimal activity, and a catalytic role, as the only available cation cofactor.
We also assayed the IN-(39-286) protein for disintegration activity. As shown in Fig. 5E, this activity was quite robust in Mn2+, as shown previously. Activity in Mg2+ was readily detectable, whereas activity was detected in Zn2+ only after prolonged exposure of the gel. Quantitation by phosphoimaging indicated that the rate of disintegration in Zn2+ was reduced by approximately 3 logarithms compared with Mn2+ and 2 logarithms compared with Mg2+ (data not shown). From these results, we conclude that Zn2+ cannot support significant disintegration activity of IN-(39-286). Interestingly, as with the full-length protein, Zn2+ also fails to support significant joining activity by IN-(39-286) (Fig. 5D).
Many enzymes active in the nucleic acid metabolism have an absolute requirement for divalent cations. However, details of the arrangements of such cations have been reported in only a few of the published structures. In the case of ASV IN, we previously identified binding of Mn2+ and Mg2+ to a single site between Asp-64 and Asp-121 (site I). Here we report the binding of two zinc ions and two cadmium ions to sites I and II. Site II ligands are Asp-64 and Glu-157, suggesting a role for this third member of the invariant triad in the D,D(35)E motif. Two more Zn2+ cations are located away from the active site of the enzyme. As it is not yet known if these sites can be occupied in the full-length protein, the significance of this binding cannot be evaluated. It is possible that metal ions bound to one or both of these sites could play a role in structural stabilization and/or activity control, as reported for other enzymes (21).
It is not certain why the complexes with different divalent cations show differential occupancy of site II. Each of the two sites contains two direct links to the protein through carboxyl oxygens, but in all structures of the isolated catalytic domain, the temperature factors for Glu-157 are considerably higher than for the two aspartates in the active site, indicating higher mobility of that side chain. This may cause more difficulty for binding of the octahedrally coordinated Mg2+, Mn2+, or Ca2+ than the tetrahedrally coordinated Zn2+, which has less stringent requirements for the geometry of its binding site and a lower preferred coordination number (22). This does not explain why the octahedrally coordinated Cd2+ would bind in site II, but we note that this ion is able to utilize both oxygens of Glu-157 for its binding, and this might enhance its stability.
The structural features of zinc ions in the active site agree well with the description of other co-catalytic sites in multi-Zn2+ or Mg2+ plus Zn2+ enzymes discussed by Vallee and Auld (23). As is commonly the case, both ions are close to each other (the distance is 3.62 Å in the high occupancy structure), and they are bridged by the carboxylate group of an aspartic acid (e.g. Asp-64). Similar arrangements have been reported in the past for other enzymes, such as phospholipase C (24) and nuclease P1 (25). In common with these structures, there is also indication of a shared water molecule bridging the two cations, although this putative water is not well ordered in ASV IN.
Active Sites of Polynucleotidyltransferases Bear Considerable SimilarityIn the absence of a bound DNA substrate, the current
structural data do not allow us to propose any specific model
concerning metal binding to site II and the mechanism by which
hydrolysis or nucleotidyl transfer occurs with ASV IN. Yang and Steitz
(14) have noted the similarity of the cluster of acidic residues
forming the active sites of enzymes in the structural superfamily to
which integrase belongs, and more detailed comparisons of metal
complexes of some of the members of this family were presented by
Bujacz et al. (12). The binding of two metal ions in the ASV
IN active site reported here invites comparison with the 3-5
exonuclease domain of the E. coli DNA polymerase I (Klenow
fragment) in which two metals are proposed to cooperate to activate an
attacking hydroxide and stabilize a pentacoordinate DNA phosphate
transition state in the active site (14, 15). In Fig. 6,
we compare metal complexes of the active sites of ASV IN, HIV-1 reverse
transcriptase RNase H, and the exonuclease domain of the Klenow
fragment of DNA polymerase I (15). Despite significant differences in
topologies of the three proteins, the similarity in positions of both
divalent cations and the coordinating carboxylates is apparent. In
this alignment, site I of ASV IN is superimposed on site A of the
Klenow fragment, both of which have been shown to have similar metal binding properties in terms of occupancy and metal ligand geometry. Alignment of site I of ASV IN with site B of the exonuclease domain does not yield as good a superposition of the coordinating
residues.
Some differences between these enzymes are also clear. When a mixture of divalent cations is present, site A in the Klenow fragment is occupied by a Zn2+ ion, whereas site B is occupied by Mg2+, with clear differences in the octahedral versus tetrahedral coordination. This is not the case for integrase, because Zn2+ clearly is preferred in both sites under higher metal concentrations, both with tetrahedral coordination. As there are only two carboxylate oxygens binding each ion, there are no geometric constraints that favor or necessitate one type of coordination. These differences may be due to the absence of coordinating ligands contributed by other portions of ASV IN or the DNA substrate. For both integrase and exonuclease, the concentration of Zn2+ necessary to observe binding is more than an order of magnitude lower than that for Mg2+. Active sites that contain more than one type of divalent cation are observed in other Zn2+-containing enzymes that act upon phosphate esters. The second metal ion is often Mg2+ as, for example, in phospholipase C or alkaline phosphatase (24, 25).
It is important to note that active site configurations different from
those shown in Fig. 6 are observed for other enzymes with
DNA-processing or polymerizing activity. Two divalent cations are
reported in one of the structures of rat polymerase (26), separated
by about 3.7 Å and also coordinated by three acidic residues, yet we
could obtain no convincing superposition on the active site of
integrase. Even more different is the active site of phage T4 RNase H
(27) where the two Mg2+ ions are 6.3 Å apart, with one of
them coordinated by only a single carboxylate oxygen and five
waters molecules, whereas the other is surrounded by six water
molecules and does not make direct contacts with any protein atom.
These examples show the limits of the comparisons and are a caveat
against drawing conclusions that might be too far-reaching.
Our biochemical analyses show that although Zn2+ is less effective than Mn2+, it can serve as a cofactor for nicking activity of the isolated catalytic core domain of ASV IN, whereas there is no detectable activity with Mg2+, Ca2+, or Cd2+. Comparisons of nicking activities in mixtures of Mn2+ and the other cations suggest that both Zn2+ and Cd2+ bind with higher affinity than Mn2+ and that Mg2+ and Ca2+ bind with lower affinity. This is consistent with the occupancies of these metals observed in our structural analyses of the catalytic domain. We also observed a sharp peak for the optimal concentration of Zn2+ at 2 mM; higher concentrations were inhibitory. The reason for this decreased activity at higher Zn2+ concentrations is not yet apparent. However, the most striking result from these studies was the observation that Zn2+ supported the endonuclease activity of the catalytic domain, whereas Mg2+, the presumed physiologically relevant cation, did not. This observation prompted us to ask whether Zn2+ or any of the other catalytic domain binding cations could serve as cofactors for processing and joining by the full-length enzyme.
As ASV IN can perform both processing and joining in vitro, in the absence of the N-terminal domain it is possible to separate the presumed structural role of Zn2+ bound to the N-terminal domain from a catalytic role. We found that both full-length ASV IN and IN-(39-286), which lacks the N-terminal Zn2+ binding motif, display both Mg2+- and Zn2+-dependent endonucleolytic processing activity. The Mg2+-dependent activity is somewhat higher with the full-length protein than with IN-(39-286), indicating that the N-terminal domain is required for optimal activity. Most importantly, both full-length and IN-(39-286) showed Zn2+-dependent processing activity almost equal to that observed with Mg2+. Other investigators have reported that the addition of Zn2+ can enhance the Mg2+-dependent activity of HIV-1 IN (6, 7). However, in their studies, stimulation was shown to be mediated by the N-terminal zinc binding domain. Thus, ours is the first report that Zn2+ can also act as a cofactor for catalysis by a retroviral integrase.
In contrast to results with processing, we could detect no joining activity by either full-length ASV IN or IN-(39-286) in the presence of Zn2+. We also observed that Zn2+ fails to support significant disintegration activity by either the catalytic core domain or IN-(39-286). Thus, the two reactions in which the nucleophile is derived from a DNA moiety and contact with "target" DNA sequences is required (i.e. joining and disintegration) are greatly impaired with Zn2+ as a cofactor. As the same triad of acidic amino acids in the catalytic center is essential for both processing and joining in the presence of Mn2+ and Mg2+ and these steps employ similar chemistry, there is no obvious explanation for this difference. It is possible that the lower preferred coordinating number of Zn2+ compared with Mn2+ or Mg2+ disfavors interactions with target DNAs or links to the protein that affect target DNA binding. Further structural analyses should help us to understand the basis for these distinct metal preferences. Lastly, the fact that Zn2+ binds tightly to the active site but can only support one step in integration may be relevant to the design of active site inhibitors of retroviral integrases.
The atomic coordinates and structure factors (codes 1VSI, 1VSH, and 1VSJ) have been deposited with the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We acknowledge the participation of M. Jaskólski in the initial stages of this project. We thank D. Matthews, L. Beese, and R. Almassy for the unpublished coordinates of proteins and G. Palm for the computer program for multiple alignment of the coordinate sets.