Limits...
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.

Show MeSH

Related in: MedlinePlus

Intermolecular crosslinking. LC-MS/MS peptide map profile obtained of the dimer crosslinked gel band of N-terminal domain of Mo-MLV integrase. The dimer was digested with 0.8 μg of trypsin at 37°C overnight. The image shows the ionization and sequence of the LC peak (indicated in the inset) corresponding at the intermolecular peptide 67–73 and 86–95 with K68 and K88 crosslinked with BS3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750625&req=5

Figure 8: Intermolecular crosslinking. LC-MS/MS peptide map profile obtained of the dimer crosslinked gel band of N-terminal domain of Mo-MLV integrase. The dimer was digested with 0.8 μg of trypsin at 37°C overnight. The image shows the ionization and sequence of the LC peak (indicated in the inset) corresponding at the intermolecular peptide 67–73 and 86–95 with K68 and K88 crosslinked with BS3.

Mentions: The next step was to determine the contact zones of the dimeric protein-protein interface, using the information of the reaction products in which the crosslinked functional groups belonged to different polypeptides (see Table 2). Figure 6 shows the profile of one of these differential peptides obtained after digestion of the dimeric band with chymotrypsin. The peptide was sequenced yielding the sequence TVTDIKDLTK24LGAIY – TK24L, which contain the crosslinked residues K24 and K24 suggesting the proximity between both N-terminal ends of the polypeptides. When a similar analysis of the crosslinked protein was carried out using trypsin, a large peptide (m/z nearly 5,000) was observed (Figure 7A) that would correspond to crosslinking between K31 and either K20 or K24 indicated for the mass of the fragment (bioworks software analysis) of 4999.5366 m/z. This peptide could not be sequenced by MS/MS due to its large size. However when the crosslinked IN 1–105 dimer was digested with chymotrypsin, a peptide corresponding to crosslinking of residues K20 and K31 on each of the polypeptide chains was identified and sequenced (Figure 7B). These results indicate that crosslinking occurs between K20 and K31 of different polypeptides. A third crosslinked peptide was identified after tryptic digestion of the crosslinked dimer in the MS/MS sequences as MK68ALLER-TLK88NITETCK indicating that K68 and K88 are crosslinked (Figure 8). The intermolecular crosslinking pattern identified shows a particular distribution along the NTD (see Table 2). These results allow us to produce a 3D model and characterize the dimeric interface.


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)

Intermolecular crosslinking. LC-MS/MS peptide map profile obtained of the dimer crosslinked gel band of N-terminal domain of Mo-MLV integrase. The dimer was digested with 0.8 μg of trypsin at 37°C overnight. The image shows the ionization and sequence of the LC peak (indicated in the inset) corresponding at the intermolecular peptide 67–73 and 86–95 with K68 and K88 crosslinked with BS3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Intermolecular crosslinking. LC-MS/MS peptide map profile obtained of the dimer crosslinked gel band of N-terminal domain of Mo-MLV integrase. The dimer was digested with 0.8 μg of trypsin at 37°C overnight. The image shows the ionization and sequence of the LC peak (indicated in the inset) corresponding at the intermolecular peptide 67–73 and 86–95 with K68 and K88 crosslinked with BS3.
Mentions: The next step was to determine the contact zones of the dimeric protein-protein interface, using the information of the reaction products in which the crosslinked functional groups belonged to different polypeptides (see Table 2). Figure 6 shows the profile of one of these differential peptides obtained after digestion of the dimeric band with chymotrypsin. The peptide was sequenced yielding the sequence TVTDIKDLTK24LGAIY – TK24L, which contain the crosslinked residues K24 and K24 suggesting the proximity between both N-terminal ends of the polypeptides. When a similar analysis of the crosslinked protein was carried out using trypsin, a large peptide (m/z nearly 5,000) was observed (Figure 7A) that would correspond to crosslinking between K31 and either K20 or K24 indicated for the mass of the fragment (bioworks software analysis) of 4999.5366 m/z. This peptide could not be sequenced by MS/MS due to its large size. However when the crosslinked IN 1–105 dimer was digested with chymotrypsin, a peptide corresponding to crosslinking of residues K20 and K31 on each of the polypeptide chains was identified and sequenced (Figure 7B). These results indicate that crosslinking occurs between K20 and K31 of different polypeptides. A third crosslinked peptide was identified after tryptic digestion of the crosslinked dimer in the MS/MS sequences as MK68ALLER-TLK88NITETCK indicating that K68 and K88 are crosslinked (Figure 8). The intermolecular crosslinking pattern identified shows a particular distribution along the NTD (see Table 2). These results allow us to produce a 3D model and characterize the dimeric interface.

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