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Crosslinking and mass spectrometry suggest that the isolated NTD domain dimer of Moloney murine leukemia virus integrase adopts a parallel arrangement in solution.

Henriquez DR, Zhao C, Zheng H, Arbildua JJ, Acevedo ML, Roth MJ, Leon O - BMC Struct. Biol. (2013)

Bottom Line: The distances between the crosslinked lysines within the monomer are in agreement with the structure of the NTD monomer found in 3NNQ.The 3D coordinates of 3NNQ were used to derive a theoretical structure of the NTD dimer with the suite 3D-Dock, based on shape and electrostatics complementarity, and filtered with the distance restraints determined in the crosslinking experiments.The crosslinking results are consistent with the monomeric structure of NTD in 3NNQ, but for the dimer, in our model both polypeptides are oriented in parallel with each other and the contacting areas between the monomers would involve the interactions between helices 1 and helices 3 and 4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Programa de Virologia ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.

ABSTRACT

Background: Retroviral integrases (INs) catalyze the integration of viral DNA in the chromosomal DNA of the infected cell. This reaction requires the multimerization of IN to coordinate a nucleophilic attack of the 3' ends of viral DNA at two staggered phosphodiester bonds on the recipient DNA. Several models indicate that a tetramer of IN would be required for two-end concerted integration. Complementation assays have shown that the N-terminal domain (NTD) of integrase is essential for concerted integration, contributing to the formation of a multimer through protein-protein interaction. The isolated NTD of Mo-MLV integrase behave as a dimer in solution however the structure of the dimer in solution is not known.

Results: In this work, crosslinking and mass spectrometry were used to identify regions involved in the dimerization of the isolated Mo-MLV NTD. The distances between the crosslinked lysines within the monomer are in agreement with the structure of the NTD monomer found in 3NNQ. The intermolecular crosslinked peptides corresponding to Lys 20-Lys 31, Lys 24-Lys 24 and Lys 68-Lys 88 were identified. The 3D coordinates of 3NNQ were used to derive a theoretical structure of the NTD dimer with the suite 3D-Dock, based on shape and electrostatics complementarity, and filtered with the distance restraints determined in the crosslinking experiments.

Conclusions: The crosslinking results are consistent with the monomeric structure of NTD in 3NNQ, but for the dimer, in our model both polypeptides are oriented in parallel with each other and the contacting areas between the monomers would involve the interactions between helices 1 and helices 3 and 4.

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Cysteine cross-linking. The reaction was carried out in 50 mM Tris–HCl pH 7.0. 50 μM of IN 1–105 K104C in buffer without crosslinker (control lane) or in the presence of 1 mM of the bi-functional maleimide crosslinker BMOE or BM(PEG)2. The reaction was done on ice for 60 min and quenched with 10 mM DTT.
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Figure 11: Cysteine cross-linking. The reaction was carried out in 50 mM Tris–HCl pH 7.0. 50 μM of IN 1–105 K104C in buffer without crosslinker (control lane) or in the presence of 1 mM of the bi-functional maleimide crosslinker BMOE or BM(PEG)2. The reaction was done on ice for 60 min and quenched with 10 mM DTT.

Mentions: In order to test our model, we performed crosslinking studies introducing cysteine residues at position 104 in IN1-105 (K104C). This amino acid residue was chosen because the Cα of both K104 in the 3NNQ dimer is separated by 28 Å. In contrast, the distance between these residues in our model is only 10 Å. Therefore crosslinking with BMOE (8 Å) would be distinguished between both models. The mutant K104C IN1-105 was obtained by site directed mutagenesis and purified as described in Materials and Methods. This protein was able to complement a concerted integration assay that used a deletion mutant IN lacking the NTD domain (Figure 10). In addition, IN 1–105 contains two cysteine residues (C94 and 96) coordinated with zinc, that are not reactive to N-ethylmaleimide [8]. K104C IN 1–105 protein was crosslinked with BMOE and BM(PEG)2 of 8 and 14.7 Å in length, respectively. Figure 11 shows that both reagents produced crosslinked dimer of K104C IN 1–105 (lanes 2 and 3) indicating that the SH groups of cysteines are within 8 Å. The estimated crosslinking extent was 65% for BMOE and 70% for BM(PEG)2. The distance between the cysteines in the 3D coordinates of 3NNQ dimer would be more than 28 Å. Furthermore in 3NNQ both carboxyl ends are separated by interactions with helix 3 of the other monomer reducing the flexibility of the polypeptide chain at the position of K104.


Crosslinking and mass spectrometry suggest that the isolated NTD domain dimer of Moloney murine leukemia virus integrase adopts a parallel arrangement in solution.

Henriquez DR, Zhao C, Zheng H, Arbildua JJ, Acevedo ML, Roth MJ, Leon O - BMC Struct. Biol. (2013)

Cysteine cross-linking. The reaction was carried out in 50 mM Tris–HCl pH 7.0. 50 μM of IN 1–105 K104C in buffer without crosslinker (control lane) or in the presence of 1 mM of the bi-functional maleimide crosslinker BMOE or BM(PEG)2. The reaction was done on ice for 60 min and quenched with 10 mM DTT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750625&req=5

Figure 11: Cysteine cross-linking. The reaction was carried out in 50 mM Tris–HCl pH 7.0. 50 μM of IN 1–105 K104C in buffer without crosslinker (control lane) or in the presence of 1 mM of the bi-functional maleimide crosslinker BMOE or BM(PEG)2. The reaction was done on ice for 60 min and quenched with 10 mM DTT.
Mentions: In order to test our model, we performed crosslinking studies introducing cysteine residues at position 104 in IN1-105 (K104C). This amino acid residue was chosen because the Cα of both K104 in the 3NNQ dimer is separated by 28 Å. In contrast, the distance between these residues in our model is only 10 Å. Therefore crosslinking with BMOE (8 Å) would be distinguished between both models. The mutant K104C IN1-105 was obtained by site directed mutagenesis and purified as described in Materials and Methods. This protein was able to complement a concerted integration assay that used a deletion mutant IN lacking the NTD domain (Figure 10). In addition, IN 1–105 contains two cysteine residues (C94 and 96) coordinated with zinc, that are not reactive to N-ethylmaleimide [8]. K104C IN 1–105 protein was crosslinked with BMOE and BM(PEG)2 of 8 and 14.7 Å in length, respectively. Figure 11 shows that both reagents produced crosslinked dimer of K104C IN 1–105 (lanes 2 and 3) indicating that the SH groups of cysteines are within 8 Å. The estimated crosslinking extent was 65% for BMOE and 70% for BM(PEG)2. The distance between the cysteines in the 3D coordinates of 3NNQ dimer would be more than 28 Å. Furthermore in 3NNQ both carboxyl ends are separated by interactions with helix 3 of the other monomer reducing the flexibility of the polypeptide chain at the position of K104.

Bottom Line: The distances between the crosslinked lysines within the monomer are in agreement with the structure of the NTD monomer found in 3NNQ.The 3D coordinates of 3NNQ were used to derive a theoretical structure of the NTD dimer with the suite 3D-Dock, based on shape and electrostatics complementarity, and filtered with the distance restraints determined in the crosslinking experiments.The crosslinking results are consistent with the monomeric structure of NTD in 3NNQ, but for the dimer, in our model both polypeptides are oriented in parallel with each other and the contacting areas between the monomers would involve the interactions between helices 1 and helices 3 and 4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Programa de Virologia ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.

ABSTRACT

Background: Retroviral integrases (INs) catalyze the integration of viral DNA in the chromosomal DNA of the infected cell. This reaction requires the multimerization of IN to coordinate a nucleophilic attack of the 3' ends of viral DNA at two staggered phosphodiester bonds on the recipient DNA. Several models indicate that a tetramer of IN would be required for two-end concerted integration. Complementation assays have shown that the N-terminal domain (NTD) of integrase is essential for concerted integration, contributing to the formation of a multimer through protein-protein interaction. The isolated NTD of Mo-MLV integrase behave as a dimer in solution however the structure of the dimer in solution is not known.

Results: In this work, crosslinking and mass spectrometry were used to identify regions involved in the dimerization of the isolated Mo-MLV NTD. The distances between the crosslinked lysines within the monomer are in agreement with the structure of the NTD monomer found in 3NNQ. The intermolecular crosslinked peptides corresponding to Lys 20-Lys 31, Lys 24-Lys 24 and Lys 68-Lys 88 were identified. The 3D coordinates of 3NNQ were used to derive a theoretical structure of the NTD dimer with the suite 3D-Dock, based on shape and electrostatics complementarity, and filtered with the distance restraints determined in the crosslinking experiments.

Conclusions: The crosslinking results are consistent with the monomeric structure of NTD in 3NNQ, but for the dimer, in our model both polypeptides are oriented in parallel with each other and the contacting areas between the monomers would involve the interactions between helices 1 and helices 3 and 4.

Show MeSH
Related in: MedlinePlus