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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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Sequence alignment of PFV, HIV-1 and Mo-MLV NTD-IN. The secondary structure of Mo-MLV is shown below. α-helices are marked as orange cylinders, and β-strands by blue arrows. Yellow boxes: at least two identical residues, Green boxes: three identical residues. Accession codes: PFV IN, CAA68999; HIV-1 IN, AAC83550 and Mo-MLV IN, P03355.
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Figure 1: Sequence alignment of PFV, HIV-1 and Mo-MLV NTD-IN. The secondary structure of Mo-MLV is shown below. α-helices are marked as orange cylinders, and β-strands by blue arrows. Yellow boxes: at least two identical residues, Green boxes: three identical residues. Accession codes: PFV IN, CAA68999; HIV-1 IN, AAC83550 and Mo-MLV IN, P03355.

Mentions: A sequence alignment of the NTD of Mo-MLV, PFV and HIV-1 integrases is presented in Figure 1. The NTD of Mo-MLV IN is 45 amino acids larger than the NTD of HIV-1 IN [8]. PFV IN also contains an extra region of 50 amino acids before the zinc binding domain. Since no quaternary structural information for Mo-MLV IN is available, in this work we explored the use of cross-linking in order to identify lysine residues that are within the range of the cross-linking spacer within the monomer or the dimer on the NTD. Cross-linked peptides were identified and sequenced by MALDI-TOF MS/MS spectroscopy. Based on these results and the 3D coordinates available in 3NNQ, a model of the NTD dimer was built. This model suggests a parallel arrangement of the NTDs.


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)

Sequence alignment of PFV, HIV-1 and Mo-MLV NTD-IN. The secondary structure of Mo-MLV is shown below. α-helices are marked as orange cylinders, and β-strands by blue arrows. Yellow boxes: at least two identical residues, Green boxes: three identical residues. Accession codes: PFV IN, CAA68999; HIV-1 IN, AAC83550 and Mo-MLV IN, P03355.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Sequence alignment of PFV, HIV-1 and Mo-MLV NTD-IN. The secondary structure of Mo-MLV is shown below. α-helices are marked as orange cylinders, and β-strands by blue arrows. Yellow boxes: at least two identical residues, Green boxes: three identical residues. Accession codes: PFV IN, CAA68999; HIV-1 IN, AAC83550 and Mo-MLV IN, P03355.
Mentions: A sequence alignment of the NTD of Mo-MLV, PFV and HIV-1 integrases is presented in Figure 1. The NTD of Mo-MLV IN is 45 amino acids larger than the NTD of HIV-1 IN [8]. PFV IN also contains an extra region of 50 amino acids before the zinc binding domain. Since no quaternary structural information for Mo-MLV IN is available, in this work we explored the use of cross-linking in order to identify lysine residues that are within the range of the cross-linking spacer within the monomer or the dimer on the NTD. Cross-linked peptides were identified and sequenced by MALDI-TOF MS/MS spectroscopy. Based on these results and the 3D coordinates available in 3NNQ, a model of the NTD dimer was built. This model suggests a parallel arrangement of the NTDs.

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