HIV-1 Integrase: Structural Organization, Conformational Changes, and Catalysis*

ADVANCES IN VIRUS RESEARCH, VOL. 52

HIV-1 INTEGRASE: STRUCTURAL ORGANIZATION, CONFORMATIONAL CHANGES, AND CATALYSIS* Ernest Asante-Appiah and Anna Marie Skalka Institute for Cancer Research Fox Chase Cancer Center Philadelphia, Pennsylvania 191 1 1

I. Introduction 11. Structural Organization of IN A. N-Terminal Domain B. Catalytic Core Domain C. C-Terminal Domain 111. Functional Oligomer A. Intermolecular Interactions B. Protein-DNA Interactions C. Multimerization D. Structural Modeling IV. Metal-Induced Conformational Changes A. Conformation-Sensitive Monoclonal Antibodies B. Limited Proteolysis V. Structural Features That Mediate Conformational Changes in HIV-1 IN VI. Conformational Changes and Catalysis A. HIV-1 IN-Metal-DNA Complex VII. Conclusion VIII. Summary References

I. INTRODUCTION The integrase (IN) protein from retroviruses catalyzes one of the critical steps in the establishment of a viral infection: the integration of the viral DNA into the genome of the host (1-3). The protein has evolved to catalyze two separate reactions that are required to accomplish this goal, a hydrolytic reaction termed “processing”and a transesterification reaction referred to as “joining” or “strand transfer.” The repertoire of activities necessary for a complete catalytic cycle includes *This work was supported by National Institutes of Health grants CA-71515, AI40385, A140721 and institutional grant from National Institute of Health CA-06927, and also by an appropriation from the Commonwealth of Pennsylvania. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute, or any other sponsoring organization. 351

Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved. 0065-3527/99 $30.00

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the ability of the protein to recognize viral DNA ends specifically but to bind the target DNA in a sequence-independent fashion. Because the processing reaction yields a product that becomes the substrate of the subsequent joining reaction, it is also imperative that the protein hold on to the processed viral DNA ends as it moves from the cytoplasm into the nucleus, where the joining reaction takes place. In this review, we address some mechanistic aspects of viral DNA integration, focusing on the contribution of structural dynamics to catalysis, particularly with respect to HIV-1 IN. We discuss studies that elucidate the role of domain interactions, and conformational changes that appear to be necessary early in the reaction. We also point out the gaps in our current knowledge of the IN mechanism of action. Wherever possible, we have framed our discussions on the basis of the available structural models of the independent domains of IN. We have, however, avoided details of three-dimensional structures of these domains, and we refer the reader to the contributions of Esposito and Craigie in this volume, where the subject is tackled in greater depth.

11. STRUCTURAL ORGANIZATION OF IN Early attempts to understand the structural basis of viral DNA integration focused on the modular nature of the IN proteins. With the aid of recombinant DNA methodology and limited proteolysis, it soon became evident that INS comprised three independently folding domains; an N-terminal domain, a catalytic core domain, and a C-terminal domain (Fig. 10.1).Efforts to understand the role of these domains have provided some interesting insights into the contribution of structural dynamics during catalysis.

A. N-Terminal Domain The N-terminal domain spans approximately the first 50 amino acids of IN. Amino acid sequence comparison of residues in this domain revealed the presence of two histidine and two cysteine residues that were evolutionarily conserved in all retroviral INS (4). This HHCC motif was reminiscent of the canonical zinc-binding motif (in reverse orientation) that is found in several DNA-binding proteins. Biochemical and biophysical experiments showed that an isolated N-terminal domain could indeed bind zinc ions (5-7). Subsequently, it was demonstrated that the binding of zinc ions promotes tetramerization of human immunodeficiency virus-1 (HIV-1) IN in uitro, and increased catalytic

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N

catalytic core

1216 4043

1

64

116

152

212 220

50

zinc-binding helix-turn-helix domain multimerization determinants

metal cofactor-binding domain

----

tail

C

multimerization determinants

270 288

nonspecific DNA-binding SH3-like domain

----

muitimerization determinants

FIG10.1. A schematic model of HIV-1 IN. The linear model shows the three independently folding domains (N terminus, catalytic core, and C terminus) of the protein whose structures are known. The region depicted as the "tail" refers to the last 18 amino acids at the C terminus, a region that is structurally unresolved. The conserved and catalytically important residues are indicated, and the corresponding residue numbers are shown above the figure. The residue numbers that delimit the domains, and the features that describe the domains, are indicated below the figure. Multimerization determinants, as indicated, are found in all the domains.

activity of the enzyme (8,9).Results with avian sarcoma virus (ASV) IN suggest that this zinc effect may not be a general phenomenon among INS. An N-terminal truncation derivative of the avian enzyme, that is missing the entire domain, can process, and join single viral DNA ends to the same extent as the full-length enzyme (10). The structures of the N-terminal domains of HIV-1 and H N - 2 IN have been determined by nuclear magnetic resonance (NMR) (11,121. Instead of the expected ab structure of classic zinc fingers, the N-terminal domains were found to be completely &-helical(see the review by Esposito and Craigie in this volume for details).

B. Catalytic Core Domain The catalytic core domain of retroviral INS spans approximately 150 amino acids, and it is characterized by the presence of three acidic amino acids (two aspartic acids and a glutamic acid), of which the last two are separated by 35 amino acids. This D,D(35)Emotif is also found in bacterial insertion sequences and some retrotransposons (13-15). The evolutionary conservation suggested a n important role for these residues in IN function. Indeed, even conservative substitutions of

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residues of this motif greatly impair all catalytic activities in vitro (15-17). An exciting development in the past few years has been the solution of the crystal structure of the isolated catalytic core domains of HIV-1 IN (18,19) and of ASV IN (20). The structures revealed that the catalytic domains of the retroviral INS fold with similar topology, as has been predicted from biochemical studies and sequence comparisons. Of particular significance to this review was the presence of a flexible loop in the structure of the core domain of HIV-1 IN that appears t o be involved in metal-induced conformational changes (cf. Fig. 10.4; see also reviews by Esposito and Craigie, and by Wlodawer, in this volume).

C. C-Terminal Domain The C-terminal region of retroviral INS is the most divergent portion of the proteins. Amino acid sequence alignment does not reveal significant homology among the INS. Despite the lack of sequence homology, the C-terminal domains of INS have been shown to possess sequenceindependent DNA binding ability that may potentially influence the binding of the target DNA into which the viral DNA integrates. The structure of a C-terminal fragment (residues 220-270) of HIV IN has been solved by NMR (21,22).The structure resembled that of the Src homology 3 (SH3) domain found in several proteins involved in signal transduction. The presence of this structural element in a DNAmetabolizing enzyme was not predicted. A saddle-shaped groove on an exposed face of a C-terminal domain dimer structure has been proposed to be the DNA-binding locus in the C terminus of HIV IN; such a model is consistent with results from site-directed mutagenesis and DNA binding studies (14,23,24). Thus, apart from the last 18 amino acids (residues 271-288) of HIV-1 IN (depicted as the “tail” in Fig. lO.l), for which no structural data are available, the basic structural architecture of the independent domains of this IN is known. The conservation of the HHCC and D,D(35)E motifs in all retroviral INS coupled with notable amino acid sequence homologies in the N-terminal and core domains suggest that the structures of these domains may also be conserved. Of course, the size differences of domains in the various INS may introduce some structural differences. For example, Moloney murine leukemia virus (MoMLV) IN has an additional 16 amino acids in the N-terminal domain compared to HIV-1 IN when the residues of the HHCC motif are aligned (4). In contrast, because of the lack of sequence conservation

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in the C-terminal domain of retroviral INS, it is not obvious if this domain is conserved structurally. The sequence diversity in this domain may signal other functions that are encoded in this region which may vary among the INS.For example, the nuclear localization signal (NLS) of ASV IN resides at the proximal end of the C-terminal domain (25). In contrast, HIV-1 NLS sequences are located in the core domain as well as in the C-terminus (26). In MLV, there is presently no evidence of an NLS sequence in IN (27). 111. FUNCTIONAL OLIGOMER A knowledge of the functional oligomeric state of any protein goes a long way toward elucidating the mechanism that underlies the processes or phenomena it influences. Although the independently folding domains of IN have been delineated and their structures (at least for HIV-1 IN) solved, the manner in which these domains interact to form the catalytically active protein is still not understood. A major stumbling block has been the inability to establish convincingly the oligomeric state of the protein during catalysis. Hence, it has been difficult t o determine stoichiometries of ligands, and to readily quantify and interpret various interactions. Although there are strong indications that retroviral INS function as oligomers, the actual species continue to elude investigators. Several properties of the protein have contributed to this uncertainty. For example, purified recombinant integrases can exist in a dynamic equilibrium that includes monomers, dimers, tetramers, and even higher-order oligomers (28-32), and it is not yet possible to exclude any species, except perhaps the monomer (29), as catalytically inactive. Additionally, the catalytic activity of the enzyme is so low that most activity-based assays require more than stoichiometric amounts of the enzyme compared to the concentration of the substrate. Despite these problems, important clues have been obtained that may contribute to the ultimate resolution of this question.

A. Intermolecular Interactions Sedimentation analysis ofASV IN in conjunction with activity assays has revealed that the enzyme is active as a multimer, minimally a dimer (29).This observation has been corroborated by complementation studies with HIV IN. The ability to reconstitute catalytic activity of HIV-1 IN by mixing two independently inactive derivatives that carry amino acid substitutions or truncations of different domains of the

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protein indicates that minimally a dimeric IN can support catalytic activity (33,341. Because the manner in which IN derivatives complement each other is not known, the involvement of higher-order species cannot be excluded in these analyses. A model of an active dimeric IN would require that each monomeric subunit must contain separate binding sites for the viral and target DNA molecules, with a single active site catalyzing both processing and joining reactions (30).

B. Protein-DNA Interactions The active oligomeric species of IN has also been studied by investigating the protein’s interaction with the DNA substrates. Activity assays have established that IN can distinguish between viral DNA ends and other oligonucleotides (35-38); however, it remains unclear how the discrimination is accomplished. In uitro, IN binds to substrate DNAs with affinities similar to those of nonsubstrate DNAs. This has been established by a number of approaches, including ultraviolet (W) cross-linking studies (39,40), filter binding assays (41,42), Southwestern blots (23,431, and electrophoretic mobility shift assays (44). This inability t o distinguish between substrates and nonsubstrates by binding assays has confounded efforts to determine the identity of the active oligomeric species. Experiments suggest that specificity in catalysis is achieved in a complex way by the contribution of several nucleotides, both distal and proximal to the scissile bond (45,46). It appears that the metal cofactor also contributes to the interaction between IN and the substrate DNAs (47-50). However, the metal ion introduces another level of complexity because it can induce aggregation ofIN (51,521. As the dissociation constant for IN-metal ion interaction is quite high (estimated in the mMrange), the protein tends to aggregate before any meaningful interactions can be measured (our unpublished results 1997). C. Multimerization If the factors that contributed to the multimerization of IN were known, then it might be possible to exploit this knowledge to identify the active oligomeric species. A combination of biophysical analysis and mutagenesis has therefore been employed in efforts to uncover the determinants of multimerization and the domains that harbor them. The application of size-exclusion chromatography, chemical crosslinking, and protein overlay methodologies showed that determinants

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that influence multimerization in ASV IN are included mainly in the catalytic core and C-terminal domains (53).Similar observations were made with HIV-1 IN (54). However, the N-terminal domain of HIV-1 IN was also found to possess determinants of multimerization (8,9). The results of these biophysical studies have been corroborated by structural analyses of the independent domains of IN. The tertiary structures of all three isolated domains of HIV-1 IN and the crystal structure of ASV IN core domain show that each domain can dimerize (11,12,18,20-22). Thus, it is clear that determinants of multimerization are contributed by all three domains of INS. This observation and the potential contribution of interdomain interactions introduce another level of complexity in delineating what influences the multimerization of IN. The contribution of interdomain communication and conformational changes induced by the metal ion in the functional oligomer remains an active area of research.

D. Structural Modeling A major advance in structure-function studies of IN has been the solution of the three-dimensional structures of all three independently folding domains of HIV-1 IN and the crystal structure of the ASV IN core. This important development has provided the framework for an attempt to reconstruct a quaternary structure of HIV-1 IN (11).The model proposed by Cai et al. (11)suggests that the protein might function as a tetramer. Previously, a tetrameric IN was proposed on the basis of biochemical experiments (44,551. A tetrameric HIV-1 IN is also suggested by biophysical and biochemical studies with zinc ions. Sizeexclusion chromatography studies have demonstrated that addition of the “missing” zinc ion to purified recombinant “metal-free” HIV-1 IN induces tetramerization (8,9). Furthermore, the resulting tetramers were considerably more active catalytically than the zinc-free protein. A tetrameric model of active IN in which each monomeric subunit interacts with either the viral or target DNA raises an interesting specificity issue. If each monomeric subunit of the presumed symmetrical homotetramer possesses binding sites of equal affinity for both viral and target DNAs, how is the correct recombination reaction specified? One possible solution is that the binding of the metal cofactor might introduce asymmetry in the protein via conformational changes prior to substrate binding. It is also conceivable that the tetrameric oligomer is not symmetrical. A recent model, based on photo-cross-linking studies with HIV-1 IN and a DNA substrate, suggests that IN may function

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as an octamer during the concerted joining of the two viral DNA ends to a target DNA (56). In this model, only two protomers provide catalytic sites. Kinetic experiments coupled to biophysical measurements suggest that IN dimers are active (29,30). The current data therefore support both dimeric and tetrameric models of IN. Whether different oligomers are required for the processing and joining reactions is unclear. The contributions of the metal cofactor (Mg2+and Mn2+)and the substrate DNA to the oligomeric state of IN under standard assay conditions also remain unclear. Pembertonet al. (49)have estimated that in the ternary IN-metal-DNA complex there are on average 10 molecules of HIV-1 IN per 21-mer oligonucleotide viral DNA end substrate. However, because substrate oligonucleotides can serve as surrogate targets that may be bound in a sequence-independent manner, a simple relationship between IN and the oligonucleotide substrate molecules cannot be assumed. Suffice it to say that establishing the oligomeric state of the functionally active IN still remains a challenge. IV. METAL-INDUCED CONFORMATIONAL CHANGES Studies of the effect of the divalent metal cofactor on the structural organization of HIV-1 IN are providing new insight into the nature of domain interactions in this IN. By scoring for the ability to inactivate HIV-1 IN chemically with N-ethylmaleimide (NEM) (a thiol-specific reagent) in the presence or absence of divalent metal ions, Ellison et al. (51) showed that HIV-1 IN undergoes a metal-dependent structural change that results in protein aggregation. This structural change was found to affect a region in the catalytic core domain (that remains to be fully characterized) and required the presence of an intact N-terminal domain. As this interdomain interaction was necessary for catalytic activity, it is likely that interaction between the N terminus and the catalytic core domain of HIV-1 IN is functionally significant (51). In support of this interpretation, it had previously been demonstrated that removal of the N-terminal domain impairs catalytic activity of HIV-1 IN in uitro (41). As already noted, ASV IN appears to be significantly different from HIV-1 IN in this respect because its N-terminal domain does not seem to be required for catalytic activity in uitro (10,57). However, the absence of the domain does abolish viral replication in tissue culture, suggesting that the ASV IN N-terminal domain may be required for protein-protein interactions in uiuo.

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A. Conformation-Sensitive Monoclonal Antibodies

Because metal ions can induce aggregation of HIV-1 IN, detailed analysis of the structural changes that take place on their binding had been hampered. Following the discovery of two solubility-promoting substitutions, F185K and C280S, that maintain catalytic activity of HIV-1 IN i n vitro (54), it was possible to distinguish metal-dependent aggregation effects from metal-induced conformational changes (52). With the aid of this soluble, monodispersed, dimeric protein, it was shown that the catalytic core domain interacts closely with the C-terminal domain. In those studies, the addition of metal ions t o HIV-1IN impaired the ability of two anti-HIV-1 IN monoclonal antibodies (MAbs 4 and 33 with epitopes in the catalytic core and C-terminal domains, respectively) to bind HIV-1 IN. Removal of the divalent metal ions with ethylenediaminetetraacetic acid (EDTA) restored the ability of the MAbs to bind HIV-1 IN ( 5 2 ) (see Fig. 10.2). This metal-induced abrogation of HIV-1 IN binding by the MAbs did not require the presence of an intact N-terminal domain. However, the catalytic core and the C-terminal domains were both essential (52).Thus, it appears that the addition of metal ions reorganizes the catalytic core and C-terminal

20 40

0.001

Nowash

0.01

0.1

1

10

[Mn2+] mM

FIG10.2. The metal-induced change in HIV-1 IN conformation is reversible. In a n enzyme-linked immunosorbent assay (ELISA) in which HIV-1 IN is immobilized on a microtiter plate, increasing concentrations of the metal cofactor (Mn2+')prevent binding by a n anti-HIV-1 IN MAb, MAb33 (A),suggesting a metal-induced conformational change. If the divalent metal ion is removed by washing several times with buffer (Tris-buffered saline) (A), the antibody now recognizes HIV-1 IN, indicating that the phenomenon is metal dependent and reversible [for details, see Asante-Appiah and Skalka (52)l.

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domains of HIV-1 IN such that certain conformational epitopes in these domains become masked.

B. Limited Proteolysis Additional evidence for a metal-induced conformational change in HIV-1 IN was obtained from proteolysis studies. A different cleavage pattern is observed when HIV-1 IN is digested with proteinase K in the absence or presence of Mn2+ions (52). More recent studies indicate that a similar metal ion effect on HIV-1 IN is observed if trypsin is used to probe for structural changes. Thus, alternative cleavage sites become available to the proteases when Mn2+is bound. Such differences are not observed with ASV IN. Thus, by this criterion as well, the avian enzyme appears to be quite different from HIV-1 IN. It is not clear why metal ions elicit alternate proteolysis cleavage patterns in HIV-1 but not in ASV IN because (at least) the Mg2+-boundcrystal structures of the core domains of the two enzymes show a similar conformation (58-61). Whether the changes in HIV-1 IN conformation induced by the metal cofactor originate primarily from inter- or intramolecular interactions also awaits further studies (Fig. 10.3). This information will be important to the design of inhibitors against this target protein for acquired immunodeficiency syndrome (AIDS) intervention.

V. STRUCTURAL FEATURES THATMEDIATE CONFORMATIONAL CHANGES IN HIV-1 IN Structural studies with ASV IN confirmed that residues of the D,D(35)Emotif coordinate the divalent metal ion(s) required for catalysis (58,59; Wlodawer, this volume). The crystal structures provided an explanation for the inability of proteins carrying substitutions in the residues of the D,D(35)Emotif t o perform any catalytic activities. Work in our laboratory has revealed that the metal-induced conformational changes in HIV-1 IN require an intact D,D(35)Emotif(62). Substituting Asp64 with either alanine or asparagine eliminated both the metalinduced conformational change and catalytic activity in HIV-1 IN. These changes were monitored with the aid of mass spectrometry by comparing tryptic digests ofan HIV-1 IN derivative that carries a D64N substitution (and is incapable of undergoing metal-induced conformational changes) with a parental derivative, HIV-1 IN F185K,C280S, that does. Of particular note, among the changes that ensue on metal

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*Ih

361

intermolecular

.

intramolecular

Metal

1

FIG10.3. A schematic showing two potential modes of metal-induced conformational changes in HIV-1IN. In (A) the change in protein conformation is due primarily to intermolecular interactions, whereas in (B)intramolecular interactions are responsible for the structural changes. It is poesible that both types of interactions may contribute to the metal-induced phenomenon. The open oval represents the catalytic core domain, the striped oval the C-terminal domain.

binding is the protection from proteolysis of an extended loop (residues 137-156) and a flanking region (residues 161-173) seen in the crystal structure of the apo core domain of HIV-1 IN (Fig. 10.4, color plate) (63-65). The loop encompasses Glu-152, a member of the D,D(35)E motif that is expected to undergo significant movement in order to aid in the coordination of a metal ion (19). Significantly, the epitope of one

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of the conformation-sensitive MAbs k e . , MAb4), which loses recognition of HIV-1 IN in the presence of metal ions, spans this region of the core domain (52). The regions affected by the conformational change also include Lys-156 and Lys-159 in the core domain, residues that have been implicated in substrate DNA binding (66). Hence, the metalinduced changes appear to recruit critical elements of the catalytic machinery of HIV-1 IN into a favorable conformation.

VI. CONFORMATIONAL CHANGES AND CATALYSIS Studies with metal ions have shown that when HIV-1 IN is preincubated with its metal cofactor, the activity of the enzyme becomes significantly greater (at least five-fold) than that of a protein sample that has not been pretreated with metal ions (48,47,52).Although this enhancement of catalytic activity was first attributed to protein aggregation (48), we know now that a similar response is seen with a nonaggregating derivative of the enzyme, HIV-1 IN F185K,C280S (52). The metal-induced enhancement of catalytic activity can be blocked by conformation-sensitive MAbs only if the antibody is added to HIV-1 IN prior t o the metal pretreatment (52). Furthermore, the antibodies can stabilize apo-HIV-1IN against metal-induced conformational changes. These observations suggested that the apo conformation of HIV-1 IN is inactive and that the protein is activated via conformational changes induced by divalent metal ion(s). It follows, therefore, that this change must precede the chemical steps of the reaction. Because the antibodysensitive conformational changes involve the catalytic core and C-terminal domains, it seems possible that in order to promote catalysis, the metal-induced structural reorganization may position the sequence-independent DNA-binding region in the C-terminal domain close to the core domain that retains sequence-specific DNA binding determinants. This intriguing hypothesis remains to be confirmed.

A. H N - 1 IN-Metal-DNA Complex Because IN can interact with DNA in a sequence-independent fashion (14,23),the issue of how the enzyme recognizes the viral DNA ends specifically continues to baffle researchers. Furthermore, at least in oitro, IN interacts with viral and potential target DNAs with similar affinities. Studies on the influence ofthe metal cofactor on DNA binding are providing some insights. Although IN can bind viral DNA in the absence of metal ions, the presence of the cation facilitates the forma-

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tion of the ternary protein-DNA-metal complex (23,40,49,51).Indeed, the preference for Mn2+over Mg2+as a metal cofactor in in uitro catalytic assays has been ascribed to the ability of the cation to promote a more stable ternary IN-metal-DNA complex (49). Interestingly, Mn2+can also induce a change in conformation in HIV-1 IN at concentrations that are an order of magnitude lower than that required to induce a similar response with Mg2’ (52). Our recent analyses show that the metal cofactor contributes to substrate discrimination by modulating the binding constants (Kd)of both viral and potential target DNA (our unpublished data; Yi Jim, Asante-Appiah, and Skalka, 1998). The involvement of the metal ion is apparently manifested in two steps. The metal cofactor facilitates the assembly of a stable IN-metalDNA complex via conformational changes in the protein and subsequently promotes catalysis. The formation of a stable complex has been characterized for the processing and joining activities of HIV-1 IN (47,51,50).For the joining reaction, it is interesting that the 5’ overhang dinucleotide that remains following the processing reaction is crucial to the formation of a stable IN-metal-processed DNA complex (48). Further studies have shown that a stable complex can be formed by a variety of divalent cations including Ca2+,which itself is unable to support catalytic activity (67). Following the formation of the stable complex, however, only a subset of divalent cations including Co2+, Mn2+,and Mg2+can serve the metal cofactor role as an electrophilic catalyst (67). It is not known if the metal cofactor remains bound to the enzyme after each catalytic cycle or if IN has to be “reprimed” before each cycle. VII. CONCLUSION Our current knowledge of the mechanistic aspects of viral DNA integration suggest a strong contribution from protein dynamics even in the earliest steps in the reaction. In the case of HIV-1 IN, the metal cofactor induces a conformational change in the enzyme that appears to reorganize regions in the core and the C-terminal domains. These changes appear to activate the enzyme by recruiting the catalytic machinery to a favorable structure. Although IN can bind viral DNA in the absence of a metal cofactor, it appears that the metal ion promotes this interaction. The IN-metal-DNA complex that forms (i.e., the stable ternary complex) is resistant to challenge by competing DNAs. Following the formation of the stable complex, the metal ion promotes the chemical steps presumably by acting as an electrophilic catalyst.

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Although it seems likely, it is not known if conformational changes play a significant role at later steps in the reaction, proceeding from processing to joining, and finally to release of the product. In this review, we have focused primarily on protein structural changes that precede catalysis. This is not meant to imply that structural changes are not induced by the substrate DNAs. Such substrate effects are beyond the scope of this review but have been addressed in considerable detail elsewhere (see review by M. Katzman and R. Katz in this volume). We anticipate that both types of structural dynamics (protein and substrate mediated) figure significantly in processing and joining viral DNA ends to the target DNA.

Integrase comprises three domains capable of folding independently and whose three-dimensional structures are known. However, the manner in which the N-terminal, catalytic core, and C-terminal domains interact in the holoenzyme remains obscure. Catalytically active recombinant IN can exist in a dynamic equilibrium of monomers, dimers, tetramers, and higher order species. Numerous studies indicate that the enzyme functions as a multimer, minimally a dimer. The IN proteins from HIV-1 and ASV have been studied most carefully with respect to the structural basis of catalysis. Although the active site of ASV IN does not undergo significant conformational changes on binding the required metal cofactor, that of HIV-1 IN does. The reversible, metal-induced conformational change in HIV-1 IN impairs the binding of some anti-HIV-1 IN monoclonal antibodies to the enzyme and results in differential susceptibility of the protein to proteolysis. This active site-mediated conformational change reorganizes the catalytic core and C-terminal domains and appears to promote an interaction that is favorable for catalysis. Other metal-dependent structural changes in HIV-1 IN include the promotion of interactions between the N terminal and the catalytic core domains and the induction of tetramers by zinc ions. The end result of these metal-induced changes is apparently the induction of an activated holoenzyme that can form a stable ternary integrase-metal-DNA complex. These structural changes, which appear to be crucial for optimum catalysis in HIV-1 IN, do not occur in ASV IN. The structural changes observed in HIV-1 IN may serve to recruit the catalytic machinery in this enzyme to a conformation that is native for ASV IN.

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ACKNOWLEDGMENTS This review would not have been possible without the outstanding contributions and insights from numerous authors over the years. We would like to apologize to all those authors whose work on this subject we have not been able to cite owing to space constraints. We would also like to thank Drs. Mark Andrake, Rich Katz, and Glenn Rall for their helpful comments and critical reading of the manuscript. We thank Dr. Andrake for help in creating Figure 10.4.

REFERENCES 1. Asante-Appiah, E., and Skalka, A. M. (1997). Molecular mechanisms in retrovirus DNA integration. Antiviral Res. 36, 139-152. 2. Katz, R. A., and Skalka, A. M. (1994). The retroviral enzymes. Annu. Reu. Biochem. 63, 133-173. 3. Goff, S . P. (1992). Genetics of retroviral integration. Annu. Rev. Genet. 26,527-544. 4. Johnson, M. S., McClure, M. A., Feng, D.-F., Gray, J., and Doolittle, R. F. (1986). Computer analysis of retroviral pol genes: Assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc. Natl. Acad. Sci. U.S.A.83, 7648-7652. 5. McEuen, A. R., Edwards, B., Koepke, K. A., Ball, A. E., Wolstenholme, A. J., Danson, M. J., and Hough, D. W. (1992). Zinc binding by retroviral integrase. Biochem. Biophys. Res. Commun. 189, 813-818. 6. Bushman, F. D., Engelman, A., Palmer, I., Singfield, P., and Craigie, R. (1993). Domains ofthe integrase protein ofhuman immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding. Proc. Natl. Acad. Sci. U.S.A.90,34283432. 7. Burke, C. J., Sanyal, G., Bruner, M. W., Ryan, J. A., LaFemina, R. L., Robbins, H. L., Zeft, A. S., Middaugh, C. R., and Cordingley, M. G. (1992). Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase. J. Biol. Chem. 267, 9639-9644. 8. Zheng, R., Jenkins, T. M., and Craigie, R. (1996). Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization and enhances activity. Proc. Natl. Acad. Sci. U.S.A. 93, 13659-13664. 9. Lee, S. P., Xiao, J.,Knutson, J. R., Lewis, M. S., and Han, M. K. (1997). ZnZ+promotes the self-association of human immunodeficiency virus type-1 integrase in uitro. Biochemistry 36, 173-180. 10. Katz, R. A., Merkel, G., and Skalka, A. M. (1996).Targeting of retroviral integrase by a fusion to a heterologous DNA binding domain: In uitro activities and incorporation of a fusion protein into viral particles. Virology 217, 178-190. 11. Cai, M., Zheng, R., Cafkey, R., Clore, M., and Gronenborn, A. M. (1997). Solution structure of N-terminal zinc binding domain of HIV-integrase. Nat. S t r u t . Bwl. 4, 567-577. 12. Eijkelenboom, A. P. A. M., van den Ent, F., Vos, A., Doreheijers, J. F., Hard, K., Tulrus, T. D., Plasterk, R. H. A., Kaptien, R., and Boelens, R. (1997). The solution structure of the amino terminal HHCC domain of HIV-2 integrase: A three-helix bundle stabilized by zinc. Cum. Biol. 7 , 739-746. 13. Fayet, O., Ramond, P., Polard, P., P d r e , M. F., and Chandler, M. (1990). Functional similarities between retroviruses and the IS3 family ofbacterial insertion sequences? Mol. Microbwl. 4, 1771-1777.

366

ERNEST ASANTE-APPIAH AND ANNA MARIE SKALKA

14. Khan, E., Mack, J. P. G., Katz, R. A., Kulkosky, J., and Skalka, A. M. (1991). Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 19, 851-860. 15. Kulkosky, J., Jones, K. S., Katz, R. A,, Mack, J. P. G., and Skalka, A. M. (1992). Residues critical for retroviral integrative recombination in a region that is highly conserved among retrovirallretrotransposon integrases and bacterial insertion sequence transposases. Mol. Cell. Biol. 12, 2331-2338. 16. Drelich, M., Wilhelm, R., and Mous, J. (1992). Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in uitro. Virology 188, 459-468. 17. Engelman, A,, and Craigie, R. (1992). Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in uitro. J. Virol. 66,6361-6369. 18. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., and Davies, D. R. (1994). Crystal structure of the catalytic domain of HIV-1 integrase: Similarity to other polynucleotidyl transferases. Science 266, 1981-1986. 19. Bujacz, G.,Alexandratos, J., Qing, Z. L., Clement-Mella, C., andWlodawer,A. (1996). The catalytic domain of human immunodeficiency virus integrase: Ordered active site in the F185H mutant. FEBS Lett. 398, 175-178. 20. Bujacz, G., Jask6lski, M., Alexandratos, J., Wlodawer, A., Merkel, G., Katz, R. A,, and Skalka, A. M. (1995). High resolution structure of the catalytic domain of avian sarcoma virus integrase. J. Mol. Biol. 263, 333-346. 21. Lodi, P. J., Ernst, J . A,, Kuszewski, J., Hickman, A. B., Engelman, A., Craigie, R., Clore, G. M., and Gronenborn, A. M. (1995). Solution structure of the DNA-binding domain of HIV-1 integrase. Biochemistry 34, 9826-9833. 22. Eijkelenboom,A. P. A. M., Puras-Lutzke, R., Boelens, R., Plasterk, R. H. A,, Kaptien, R., and Hitrd, K. (1995). The DNA binding domain of HIV-1 integrase has an SH3like fold. Nut. Struct. Biol. 2, 807-810. 23. Vink, C., Groeneger, A. A. M. O., and Plasterk, R. H. A. (1993). Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type 1 integrase protein. Nucleic Acids Res. 21, 1419-1425. 24. Puras-Lutzke, R., Vink, C., and Plasterk, R. H. A. (1994). Characterization of the minimal DNA-binding domain of the HIV integrase protein. Nucleic Acids Res. 22, 4125-4131. 25. Kukolj, G., Jones, K. S., and Skalka, A. M. (1997). Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases. J . Virol. 71, 843-847. 26. Gallay, P., Hope, T., Chin, D., and Trono, D. (1997). HIV-1infection of nondividing cells through the recognition of integrase by the importinkaryopherin pathway. Proc. Natl. Acad. Sci. U.S.A. 94, 9825-9830. 27. Roe, T., Reynolds, T. C., Yu, G., and Brown, P. 0. (1993). Integration of murine leukaemia virus depends on mitosis. EMBO J. 12, 2099-2108. 28. Sherman, P. A,, and Fyfe, J. A. (1990). Human immunodeficiency virus integration protein expressed in Escherichiu coli possesses selective DNA cleaving activity. Proc. Natl. Acud. Sci. U.S.A. 87, 5119-5123. 29. Jones, K. S., Coleman, J., Merkel, G. W., Laue, T. M., and Skalka, A. M. (1992). Retroviral integrase functions as a multimer and can turn over catalytically. J . Biol. Chem. 267, 16037-16040. 30. Vincent, K. A,, Ellison, V., Chow, S. A., and Brown, P. 0. (1993). Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations. J. Virol. 67, 425-437.

IN: STRUCTURAL CHANGES AND CATALYSIS

367

31. Grandgenett, D. P.,and Goodarzi, G. (1994).Folding of the multidomain human immunodeficiency virus type-1 integrase. Protein Sci. 3, 888-897. 32. Asante-Appiah, E., Merkel, G., and Skalka, A. M. (1998).Purification of untagged retroviral integrases by immobilized metal ion affinity chromatography. Protein Express Purif: 12, 105-110. 33. van Gent, D. C., Vink, C., Groeneger, A. A. M. O., and Plasterk, R. H. A. (1993). Complementation between HIV integrase proteins mutated in different domains. EMBO J. 12,3261-3268. 34. Engelman, A., Bushman, F. D. and Craigie, R. (1993).Identification of discrete functional domains of HIV-1integrase and their organization within an active multimenc complex. EMBO J. 12,3269-3275. 35. Bushman, F. D., and Craigie, R. (1991).Activities of human immunodeficiency virus (HIV) integration protein in uitro: Specific cleavage and integration of HIV DNA. Proc. Natl. Acad. Sci. U.S.A.88, 1339-1343. 36. Vink, C., van Gent, D. C., Elgersma, Y., and Plasterk, R. H. A. (1991).Human immunodeficiency virus type 1 integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J. Virol. 65, 4636-4644. 37. LaFemina, R. L.,Callahan, P. L., and Cordingley, M. G. (1991).Substrate specificity of recombinant human immunodeficiency virus type 1 integrase protein. J. Virol. 65,5624-5630. 38. Sherman, P. A., Dickson, M. L., and Fyfe, J. A. (1992).Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions. J. Virol. 66, 3593-3601. 39. Roth, M. J., Tanese, N., and Goff, S. P. (1988). Gene product of Moloney murine leukaemia virus required for proviral integration is a DNA-binding protein. J. Mol. Biol. 203, 131-139. 40. Engelman, A,, Hickman, A. B., and Craigie, R. (1994).The core and C-terminal domains of the integrase from human immunodeficiency virus type 1 each contribute to nonspecific DNA binding. J. Virol. 68, 5911-5917. 41. Schaeur, M., and Billich, A. (1992). The N-terminal region of HIV-1integrase is required for integration activity, but not for DNA binding. Biochem. Biophys. Res. Commun. 185, 874-880. 42. Mdler, B.,Bizub-Bender, D., Andrake, M., Jones, K. S., and Skalka, A. M.(1995). Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro. J. Virol. 69,5631-5639. 43. Woerner, A. M., and Marcus-Sekura, C. J. (1993).Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. Nucleic Acids Res. 21, 3507-3511. 44. Hazuda, D. J.,Wolfe, A. L., Hastings, J. C., Robbins, H. L., Graham, P. L., LaFemina, R. L., and Emini, E. A. (1994).Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase. J. Biol. Chem. 269, 3999-4004. 45. Hong, T., Murphy, E., Groarke, J., and Drlica, K. (1993).Human immunodeficiency virus type 1 DNA integration: Fine structure target analysis using synthetic oligonucleotides. J. Virol. 67,1127-1131. 46. Katzman, M., and Sudol, M. (1996).Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases. J. Virol. 70,9069-9073. 47. Vink, C., Lutzke, R. A,, and Plasterk, R. H. (1994).Formation of a stable complex between the human immunodeficiencyvirus integrase protein and viral DNA. Nucleic Acids Res. 22, 4103-4110.

368

ERNEST ASANTE-APPIAH AND ANNA MARIE SKALKA

48. Ellison, V., and Brown, P. 0. (1994).A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in uitro. Proc. Natl. Acad. Sci. U.S.A. 91,7316-7320. 49. Pemberton, I. K.,Buckle, M., and Buc, H. (1996).The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits preference for Mn(I1)rather than Mg(I1). J. Biol. Chem. 271, 1498-1506. 50. Wolfe, A. L.,Felock, P. J., Hastings, J . C., Blau, C. U., and Hazuda, D. J . (1996). The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates. J. Virol. 70, 1424-1432. 51. Ellison, V.,Gerton, J., Vincent, K. A., and Brown, P. 0. (1995).An essential interaction between distinct domains of HIV-1integrase mediates assembly of the active multimer. J. Biol. Chem. 270, 3320-3326. 52. Asante-Appiah, E., and Skalka, A. M. (1997).A metal-induced conformational change and activation in HIV-1integrase. J. Biol. Chem. 272, 16196-16205. 53. Andrake, M., and Skalka, A. M. (1995).Multimerization determinants reside in both the catalytic core and C-terminus of avian sarcoma virus integrase. J. Biol. Chem. 270,29299-29306. 54. Jenkins, T.M., Engelman, A,, Ghirlando, R., and Craigie, R. (1996).A soluble active mutant of HIV-1integrase: Multimerization involves the core and C-terminal domains. J. Biol. Chem. 271, 7712-7718. 55. Engelman, A., Mizuuchi, K., and Craigie, R. (1991).HIV-1DNA integration: Mechanism of viral DNA cleavage and DNA strand transfer. Cell (Cambridge, Mass.) 67, 1211-1221. 56. Heuer, T.S.,and Brown, P. 0. (1998).Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integraseDNA complex. Biochemistry 37,6667-6678. 57. Bushman, F. D., and Wang, B. (1994).Rous sarcoma virus integrase: Mapping functions for catalysis and substrate binding. J. Virol. 68,2215-2223. 58. Bujacz, G., Jaskblski, M., Alexandratos, J., Wlodawer, A., Merkel, G., Katz, R. A., and Skalka, A. M. (1995).The catalytic domain of avian sarcoma virus integrase: Confirmation of the active site residues in the presence of divalent cations. Structure 4,89-96. 59. Bujacz, G., Jaskblski, M., Alexandratos, J., Wlodawer, A., Merkel, G., Andrake, M. A., Katz, R. A,, and Skalka, A. M. (1997).Binding of different divalent cations to the active site of ASV integrase and their effects on enzymatic activity. J. Biol. Chem. 272, 18161-18168. 60. Maigan, S.,Guilloteau, J.-P., Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998). Crystal structures of the catalytic domain of HIV-1integrase free and complexed with its metal cofactor: High level of similarity of the active site with other viral integrases. J. Mol. Biol. 282, 359-368. 61. Goldgur, Y.,Dyda, F., Hickman, A. B., Jenkins, T. M., Craigie, R., and Davies, D. D. (1998).Three new structures of the core domain of HIV-1integrase: An active site that binds magnesium. Proc. Natl. Acad. Sci. U S A . 95,9150-9154. 62. Asante-Appiah, E., Seeholzer, S. H., and Skalka, A. M. (1998).Structural determinants of metal-induced conformational changes in HIV-1integrase. J. Bwl. Chem. 273,35078-35087. 63. Kraulis, P. J. (1991).MOLSCRIPT:Aprogram to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946-950.

IN: STRUCTURAL CHANGES AND CATALYSIS

369

64. Bacon, D. J.,and Anderson, W. F. (1988).A fast algorithm for rendering space-filling molecule pictures. J. Mol. Graphics 6,219-220. 65. Merrit, E.A.,and Murphy, M. E. P. (1994).Raster 3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr, Sect. D 50, 869-873. 66. Jenkins, T.M., Esposito, D., Engelman, A., and Craigie, R. (1997).Critical contacts between HIV-1integrase and viral DNA identified by structure-based analysis and photo-crosslinking. EMBO J. 16,6849-6859. 67. Hazuda, D. J., Felock, P. J., Hastings, J. C., Pramanik, B., and Wolfe, A. L. (1997). Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase. J. Virol. 71,7005-7011.