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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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Characterization of crosslinked product for molecular exclusion. 2 mg of IN 1–105 were crosslinked in 200 μL and loaded on a Superdex S-200 column equilibrated in 10 mM tris pH 7.5, 0.5 M NaCl, 1 mM DTT and 5% glycerol at a flow rate of 0.5 mL/min. 0.5 mL of each fraction were collected. Protein elution was monitored by the absorbance at 220 nm. The chromatographic profile of the crosslinked protein is shown in black, and unmodified control protein is shown in red. The elution position of molecular weight markers is indicated by arrows. The markers used were: β-amylase (200 kDa), serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytocrome C (12.4 kDa). The (V0) indicated the void volume (dextran blue).
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Figure 3: Characterization of crosslinked product for molecular exclusion. 2 mg of IN 1–105 were crosslinked in 200 μL and loaded on a Superdex S-200 column equilibrated in 10 mM tris pH 7.5, 0.5 M NaCl, 1 mM DTT and 5% glycerol at a flow rate of 0.5 mL/min. 0.5 mL of each fraction were collected. Protein elution was monitored by the absorbance at 220 nm. The chromatographic profile of the crosslinked protein is shown in black, and unmodified control protein is shown in red. The elution position of molecular weight markers is indicated by arrows. The markers used were: β-amylase (200 kDa), serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytocrome C (12.4 kDa). The (V0) indicated the void volume (dextran blue).

Mentions: In order to determine the size of the crosslinked protein complex, the crosslinking mixture was analyzed by gel filtration on Superdex S-200 under nondenaturing conditions. The elution profiles of crosslinked and unmodified IN 1–105 were almost identical (Figure 3), and the majority of the crosslinked IN 1–105 (~95%) elutes at the same position of the unmodified dimer. This result indicates that, under the conditions of the reaction, intermolecular crosslinking occurs within each dimer and rules out crosslinking between the dimers.


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)

Characterization of crosslinked product for molecular exclusion. 2 mg of IN 1–105 were crosslinked in 200 μL and loaded on a Superdex S-200 column equilibrated in 10 mM tris pH 7.5, 0.5 M NaCl, 1 mM DTT and 5% glycerol at a flow rate of 0.5 mL/min. 0.5 mL of each fraction were collected. Protein elution was monitored by the absorbance at 220 nm. The chromatographic profile of the crosslinked protein is shown in black, and unmodified control protein is shown in red. The elution position of molecular weight markers is indicated by arrows. The markers used were: β-amylase (200 kDa), serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytocrome C (12.4 kDa). The (V0) indicated the void volume (dextran blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Characterization of crosslinked product for molecular exclusion. 2 mg of IN 1–105 were crosslinked in 200 μL and loaded on a Superdex S-200 column equilibrated in 10 mM tris pH 7.5, 0.5 M NaCl, 1 mM DTT and 5% glycerol at a flow rate of 0.5 mL/min. 0.5 mL of each fraction were collected. Protein elution was monitored by the absorbance at 220 nm. The chromatographic profile of the crosslinked protein is shown in black, and unmodified control protein is shown in red. The elution position of molecular weight markers is indicated by arrows. The markers used were: β-amylase (200 kDa), serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytocrome C (12.4 kDa). The (V0) indicated the void volume (dextran blue).
Mentions: In order to determine the size of the crosslinked protein complex, the crosslinking mixture was analyzed by gel filtration on Superdex S-200 under nondenaturing conditions. The elution profiles of crosslinked and unmodified IN 1–105 were almost identical (Figure 3), and the majority of the crosslinked IN 1–105 (~95%) elutes at the same position of the unmodified dimer. This result indicates that, under the conditions of the reaction, intermolecular crosslinking occurs within each dimer and rules out crosslinking between the dimers.

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