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Biomedical subjects

R Craigie

Publications and source records attributed to R Craigie.

At least 19 recordsLinked to original sources
PubMed2000

Structural basis of DNA bridging by barrier-to-autointegration factor.

Barrier-to-autointegration factor (BAF) is a host cell protein that plays a crucial role in retroviral integration. Preintegration complexes (PICs) stripped of BAF lose their normal integration activity, which can be restored by incubation with purified BAF. BAF bridges double-stranded DNA both intra- and intermolecularly in a non-sequence-specific manner, leading to the formation of a nucleoprotein network. BAF also binds to the nuclear protein lamina-associated polypeptide 2 (LAP2), and is localized with chromatin during interphase and mitosis. The crystal structure of homodimeric human BAF has been determined to 1.9 A resolution. The fold of the BAF monomer resembles that of the second domain of RuvA. This comparison revealed the presence of the helix-hairpin-helix (HhH) nonspecific DNA binding motif within BAF. A novel feature of BAF's HhH motif is the occupation of the metal binding site by the epsilon-amino group of Lys 6, providing an alternative means of sequestering positive charge. Mutational analysis corroborates the HhH motif's prominent role in DNA binding and argues against a previously proposed helix-turn-helix (HTH) binding site located in another region of the monomer. A model of BAF bridging DNA via the HhH motif is proposed.

Amino Acid Sequence↗
PubMed2000

Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex.

Barrier-to-autointegration factor (BAF) is a highly conserved cellular protein that was identified by its activity in protecting retroviral DNA against autointegration. We show that BAF has the property of bridging double-stranded DNA in a highly ordered nucleoprotein complex. Whereas BAF protein alone is a dimer in solution, upon binding DNA, BAF forms a dodecamer with DNA bound at multiple discrete sites in the complex. The interactions between BAF and DNA are entirely nonspecific with respect to DNA sequence. The dual interaction of BAF with DNA and LAP2, a protein associated with the nuclear lamina, suggests a role for LAP2 in chromosome organization. Consistent with this idea, RNA interference experiments with Caenorhabditis elegans reveal a defect in mitosis.

Animals↗
PubMed1999

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design.

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Binding Sites↗
PubMed1999

HIV integrase structure and function.

HIV integrase consists of three domains, the structures of which have been individually determined by X-ray crystallography or NMR spectroscopy. The core domain, spanning residues 50-212, is responsible for the catalytic activity of the enzyme. The crystal structure of a dimer of this domain shows similarity to other proteins that carry out polynucleotidyl transfer, including MuA transposase and RNase H. The small N-terminal domain folds into a dimeric helix-turn-helix structure, which is stabilized by the coordination of zinc with conserved His and Cys residues. The function of this domain is unclear; however, it is required for integration activity and enhances tetramerization in the context of the full-length integrase. The C-terminal domain, which has an SH3-like fold, is involved in DNA binding. The structure of this domain reveals a large saddle-shaped cleft that is formed by dimerization. This cleft contains a number of positively charged residues, and its dimensions are appropriate for accommodating a double-stranded DNA helix. Although the C-terminal domain was originally believed to be involved in target DNA binding, more recent evidence suggests that it may bind to both the ends of the viral DNA and to the target DNA. Although the individual domain structures provide some insights into the function of the protein, a more detailed understanding of the complete mechanism by which integrase binds, cleaves, and transfers DNA requires a greater knowledge of how these domains are arranged in the active multimer.

Animals↗
PubMed1998

Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.

HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.

Cations↗
PubMed1998

Footprints on the viral DNA ends in moloney murine leukemia virus preintegration complexes reflect a specific association with integrase.

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3' end processing.

3T3 Cells↗
PubMed1998

Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium.

HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg2+, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg2+. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix alpha4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg2+, and could provide a satisfactory platform for binding of inhibitors.

Binding Sites↗
PubMed1998

A previously unidentified host protein protects retroviral DNA from autointegration.

Integration of a DNA copy of the viral genome into a host chromosome is an essential step in the retrovirus life cycle. The machinery that carries out the integration reaction is a nucleoprotein complex derived from the core of the infecting virion. To successfully integrate into host DNA, the viral DNA within this complex must avoid self-destructive integration into itself, a reaction termed autointegration. We have previously shown [Lee, M. S. and Craigie, R. (1994) Proc. Natl. Acad. Sci. USA 91, 9823-9827] that viral nucleoprotein complexes isolated from Moloney murine leukemia virus-infected cells exhibit a barrier to autointegration. This autointegration barrier could be destroyed by stripping factors from the complexes and subsequently restored by incubation with a host cell extract, but not by incubation with an extract of disrupted virions. We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH 3T3 fibroblasts. It is a single polypeptide of 89 aa that does not match any previously identified protein. The identity of the protein was confirmed by expressing it in Escherichia coli and demonstrating the activity of the heterologously expressed protein in the reconstitution assay.

3T3 Cells↗
PubMed1998

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.

Binding Sites↗
PubMed1998

An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.

A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.

Carbon Isotopes↗
PubMed1998

Solution structure of the cellular factor BAF responsible for protecting retroviral DNA from autointegration.

The solution structure of the human barrier-to-autointegration factor, BAF, a 21,000 Mr dimer, has been solved by NMR, including extensive use of dipolar couplings which provide a priori long range structural information. BAF is a highly evolutionarily conserved DNA binding protein that is responsible for inhibiting autointegration of retroviral DNA, thereby promoting integration of retroviral DNA into the host chromosome. BAF is largely helical, and each subunit is composed of five helices. The dimer is elongated in shape and the dimer interface comprises principally hydrophobic contacts supplemented by a single salt bridge. Despite the absence of any sequence similarity to any other known protein family, the topology of helices 3-5 is similar to that of a number of DNA binding proteins, with helices 4 and 5 constituting a helix-turn-helix motif. A model for the interaction of BAF with DNA that is consistent with structural and mutagenesis data is proposed.

Amino Acid Sequence↗
PubMed1997

A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration.

We have probed the nucleoprotein organization of Moloney murine leukemia virus (MLV) pre-integration complexes using a novel footprinting technique that utilizes a simplified in vitro phage Mu transposition system. We find that several hundred base pairs at each end of the viral DNA are organized in a large nucleoprotein complex, which we call the intasome. This structure is not formed when pre-integration complexes are made by infecting cells with integrase-minus virus, demonstrating a requirement for integrase. In contrast, footprinting of internal regions of the viral DNA did not reveal significant differences between pre-integration complexes with and without integrase. Treatment with high salt disrupts the intasome in parallel with loss of intermolecular integration activity. We show that a cellular factor is required for reconstitution of the intasome. Finally, we demonstrate that DNA-protein interactions involving extensive regions at the ends of the viral DNA are functionally important for retroviral DNA integration activity. Current in vitro integration systems utilizing purified integrase lack the full fidelity of the in vivo reaction. Our results indicate that both host factors and long viral DNA substrates may be required to reconstitute an in vitro system with all the hallmarks of DNA integration in vivo.

Animals↗
PubMed1997

Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.

Analysis of the crystal structure of HIV-1 integrase reveals a cluster of lysine residues near the active site. Using site-directed mutagenesis and photo-crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3' processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication-defective at the step of integration. Photo-crosslinking, using 5-iododeoxyuracil-substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction.

Amino Acid Sequence↗
PubMed1997

Solution structure of the N-terminal zinc binding domain of HIV-1 integrase.

The solution structure of the N-terminal zinc binding domain (residues 1-55; IN1-55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1-55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1-55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9-18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DNA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1-55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1-55 uses this helix for dimerization.

Bacterial Proteins↗
PubMed1997

Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site.

Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus. Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus. PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine. The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities. Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.

Binding Sites↗
PubMed1996

Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity.

The N-terminal domain of HIV-1 integrase contains a pair of His and Cys residues (the HHCC motif) that are conserved among retroviral integrases. Although His and Cys residues are often involved in binding zinc, the HHCC motif does not correspond to any recognized class of zinc binding domain. We have investigated the binding of zinc to HIV-1 integrase protein and find that it binds zinc with a stoichiometry of one zinc per integrase monomer. Analysis of zinc binding to deletion derivatives of integrase locates the binding site to the N-terminal domain. Integrase with a mutation in the HHCC motif does not bind zinc, consistent with coordination of zinc by these residues. The isolated N-terminal domain is disordered in the absence of zinc but, in the presence of zinc, it adopts a secondary structure with a high alpha helical content. Integrase bound by zinc tetramerizes more readily than the apoenzyme and is also more active than the apoenzyme in in vitro integration assays. We conclude that binding of zinc to the HHCC motif stabilizes the folded state of the N-terminal domain of integrase and bound zinc is required for optimal enzymatic activity.

Amino Acid Sequence↗
PubMed1996

A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization.

Structural studies of human immunodeficiency virus type 1 (HIV-1) integrase have been impeded by the low solubility of the protein. By systematic replacement of hydrophobic residues, we previously identified a single amino acid change (F185K) that dramatically improved the solubility of the catalytic domain of HIV-1 integrase and enabled the structure to be determined by x-ray crystallography. We have introduced the same mutation into full-length HIV-1 integrase. The resulting recombinant protein is soluble and fully active in vitro, whereas, HIV-1 carrying the mutation is replication-defective due to improper virus assembly. Analysis of the recombinant protein by gel filtration and sedimentation equilibrium demonstrate a dimer-tetramer self-association. We find that the regions involved in multimerization map to both the catalytic core and carboxyl-terminal domains. The dramatically improved solubility of this protein make it a good candidate for structural studies.

Amino Acid Sequence↗