Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.     

Structural analysis of a mutant of the HIV-1 integrase zinc finger domain that forms a single conformation

HIV-1 integrase consists of three functional domains, an N-terminal zinc finger domain, a catalytic core domain and a C-terminal DNA binding domain. NMR analysis of an isolated N-terminal domain (IN(1-55)) has shown that IN(1-55) exists in two conformational states [E and D forms; Cai et al. (1997) Nat. Struct. Biol. 4, 567-577]. The two forms differ in the coordination of the zinc ion by two histidine residues. In the present study, structural analysis of a mutant of IN(1-55), Y15A, by NMR spectroscopy indicated that the mutant protein folds correctly but takes only the E form. Since the Y15A mutation abrogates the HIV-1 infectivity, Y15 might have some important role in the full-length integrase activity during the virus infection cycle. Our results suggest a possible role of Y15 in structural transition between the E and D forms of HIV-1 integrase to allow the optimal tetramerization.     

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.     

Characterization of the functional domains of human foamy virus integrase using chimeric integrases

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.     

The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.     

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.     

Mapping DNA-binding sites of HIV-1 integrase by protein footprinting.

The HIV-1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full-length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full-length soluble integrase mutant, INF(185K,C280S), to proteolytic attack in the absence and presence of DNA. The N-terminal and C-terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N-terminal domain, Lys111, Lys136, Glu138, Lys156-Lys160, Lys185-Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C-terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.     

Minimal Core Domain of HIV-1 Integrase for Biological Activity

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.     

Structural and dynamical properties of a full-length HIV-1 integrase: molecular dynamics simulations

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg2+ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glu152; (iii) distances and solvation of the Mg2+; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Modeling HIV-1 integrase complexes based on their hydrodynamic properties

We present a model structure of a candidate tetramer for HIV-1 integrase. The model was built in three steps using data from fluorescence anisotropy, structures of the individual integrase domains, cross-linking data, and other biochemical data. First, the structure of the full-length integrase monomer was modeled using the individual domain structures and the hydrodynamic properties of the full-length protein that were recently measured by fluorescence depolarization. We calculated the rotational correlation times for different arrangements of three integrase domains, revealing that only structures with close proximity among the domains satisfied the experimental data. The orientations of the domains were constrained by iterative tests against the data on cross-linking and footprinting in integrase-DNA complexes. Second, the structure of an integrase dimer was obtained by joining the model monomers in accordance with the available dimeric crystal structures of the catalytic core. The hydrodynamic properties of the dimer were in agreement with the experimental values. Third, the active sites of the two model dimers were placed in agreement with the spacing between the sites of integration on target DNA as well as the integrase-DNA cross-linking data, resulting in twofold symmetry of a tetrameric complex. The model is consistent with the experimental data indicating that the F185K substitution, which is found in the model at a tetramerization interface, selectively disrupts correct complex formation in vitro and HIV replication in vivo. Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors.     

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg²⁺ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg²⁺; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Crystal structures of catalytic core domain of BIV integrase: implications for the interaction between integrase and target DNA

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45? and 2.2?, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 ?, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.     

Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily

The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.     

Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.     

Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.     

Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins

The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Diversity in the serine recombinases

Most site-specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or lambda integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N-terminal catalytic domain and a C-terminal DNA binding domain. Phage-encoded serine recombinases (e.g. phiC31 integrase) cause integration and excision with strictly controlled directionality, and have an N-terminal catalytic domain but much longer C-terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N-terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.