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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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Modeling of dimer conformation of N-terminal domain of Mo-MLV integrase. Visualization of intermolecular crosslinking products with Swiss-PdbViewer of the dimeric interfase of NTD of Mo-MLV integrase (3NNQ pdb template, experimental crosslinking strain and 3D-Dock suite used for model). The amino acid involved in the crosslinking and its distances are indicated K31-K20 (green 20.02 Å), K24-K24 (red 15.14 Å) and K88-K68 (yellow 13.57 Å), all of them are in agreement with the spacer arm of BS3. K104 (light blue) and zinc atoms (yellow) are also indicated.
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Figure 9: Modeling of dimer conformation of N-terminal domain of Mo-MLV integrase. Visualization of intermolecular crosslinking products with Swiss-PdbViewer of the dimeric interfase of NTD of Mo-MLV integrase (3NNQ pdb template, experimental crosslinking strain and 3D-Dock suite used for model). The amino acid involved in the crosslinking and its distances are indicated K31-K20 (green 20.02 Å), K24-K24 (red 15.14 Å) and K88-K68 (yellow 13.57 Å), all of them are in agreement with the spacer arm of BS3. K104 (light blue) and zinc atoms (yellow) are also indicated.

Mentions: 3D-dock suite was used to generate 10,000 possible complexes through the rotation and translation of a mobile monomer (3NNQ), around the fixed coordinates of another 3NNQ monomer. This set of possible dimers was filtered using the experimental crosslinking information, to select the complex that agrees with the distance restraint data. A distance of 21.3 Å between crosslinked lysines was used to select complex candidates. The selected dimers were submitted to an energy minimization protocol in order to optimize steric and electrostatic interactions of the residues involved in the protein - protein interface. All the dimeric structures that satisfied the distance restraints showed a parallel disposition, with the residues involved in the crosslinking distributed in the same longitudinal face of the protein and both polypeptide chains arranged in the same direction. The complex with the more stable interface, according to Multidock routine of 3D-dock suite, was chosen as the more probable NTD - NTD dimer. A ribbon representation of the selected model is presented in Figure 9, showing the lysine residues that were crosslinked. This model shows a symmetric and parallel orientation of the monomers and two points of contact. (1) In the first α-helix of the N-terminal domain (between K24-K24) and (2) the fourth α-helix of chain A (blue) with the fourth α-helix of chain B (brown) of the dimer. This parallel arrangement would be mainly stabilized by electrostatic and Van der Waals interactions, where residues: L 25, D 84, R 85 and D 92 of monomer A and K 24, L 25, D 84 and D 92 of monomer B, play the main role in complex stabilization. The complex is characterized for a ΔASA of 484 Å2, involving 16 residues of the first monomer and 17 residues of the second monomer. The gain in ΔG of solvation from the protein-protein interaction was estimated by PDBePISA server in 2.5 Kcal/Mol (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver).


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)

Modeling of dimer conformation of N-terminal domain of Mo-MLV integrase. Visualization of intermolecular crosslinking products with Swiss-PdbViewer of the dimeric interfase of NTD of Mo-MLV integrase (3NNQ pdb template, experimental crosslinking strain and 3D-Dock suite used for model). The amino acid involved in the crosslinking and its distances are indicated K31-K20 (green 20.02 Å), K24-K24 (red 15.14 Å) and K88-K68 (yellow 13.57 Å), all of them are in agreement with the spacer arm of BS3. K104 (light blue) and zinc atoms (yellow) are also indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Modeling of dimer conformation of N-terminal domain of Mo-MLV integrase. Visualization of intermolecular crosslinking products with Swiss-PdbViewer of the dimeric interfase of NTD of Mo-MLV integrase (3NNQ pdb template, experimental crosslinking strain and 3D-Dock suite used for model). The amino acid involved in the crosslinking and its distances are indicated K31-K20 (green 20.02 Å), K24-K24 (red 15.14 Å) and K88-K68 (yellow 13.57 Å), all of them are in agreement with the spacer arm of BS3. K104 (light blue) and zinc atoms (yellow) are also indicated.
Mentions: 3D-dock suite was used to generate 10,000 possible complexes through the rotation and translation of a mobile monomer (3NNQ), around the fixed coordinates of another 3NNQ monomer. This set of possible dimers was filtered using the experimental crosslinking information, to select the complex that agrees with the distance restraint data. A distance of 21.3 Å between crosslinked lysines was used to select complex candidates. The selected dimers were submitted to an energy minimization protocol in order to optimize steric and electrostatic interactions of the residues involved in the protein - protein interface. All the dimeric structures that satisfied the distance restraints showed a parallel disposition, with the residues involved in the crosslinking distributed in the same longitudinal face of the protein and both polypeptide chains arranged in the same direction. The complex with the more stable interface, according to Multidock routine of 3D-dock suite, was chosen as the more probable NTD - NTD dimer. A ribbon representation of the selected model is presented in Figure 9, showing the lysine residues that were crosslinked. This model shows a symmetric and parallel orientation of the monomers and two points of contact. (1) In the first α-helix of the N-terminal domain (between K24-K24) and (2) the fourth α-helix of chain A (blue) with the fourth α-helix of chain B (brown) of the dimer. This parallel arrangement would be mainly stabilized by electrostatic and Van der Waals interactions, where residues: L 25, D 84, R 85 and D 92 of monomer A and K 24, L 25, D 84 and D 92 of monomer B, play the main role in complex stabilization. The complex is characterized for a ΔASA of 484 Å2, involving 16 residues of the first monomer and 17 residues of the second monomer. The gain in ΔG of solvation from the protein-protein interaction was estimated by PDBePISA server in 2.5 Kcal/Mol (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver).

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