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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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LC-MS/MS peptide map profile of N-terminal domain of Mo-MLV integrase monomer crosslinked gel band digested with trypsin. (A), in the sample the peak corresponds to the peptide 86–105 with two cross-linked lysines (K88 and K95). (B), the peak corresponds at the peptide 25–34 with intramolecular cross-linked between K31 and K33, (C) in the monomeric sample the peak corresponds at the peptide 89–105 with intramolecular cross-linked between K95 and K104.
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Figure 4: LC-MS/MS peptide map profile of N-terminal domain of Mo-MLV integrase monomer crosslinked gel band digested with trypsin. (A), in the sample the peak corresponds to the peptide 86–105 with two cross-linked lysines (K88 and K95). (B), the peak corresponds at the peptide 25–34 with intramolecular cross-linked between K31 and K33, (C) in the monomeric sample the peak corresponds at the peptide 89–105 with intramolecular cross-linked between K95 and K104.

Mentions: Chemical crosslinking combined with mass spectrometry (MS) provides a useful method for inferring sites of protein-protein interactions and for mapping the topology of protein complexes. We used the bi-functional crosslinking reagent BS3 that reacts with primary amine groups of lysines and the NTD integrase of Mo-MLV. In these experiments IN 1–105 was crosslinked with BS3 and the dimeric and monomeric band was separated by SDS-PAGE, subjected to digestion with trypsin or chymotrypsin and analyzed by LC-MS/MS as indicated in Materials and Methods, being compared with the MS/MS profile of the unmodified IN 1–105, sequencing only differential peaks (Tables 1 and 2). In these cases only two informative sequences were used and classified as (1) intramolecular crosslinked peptides (or looplinks) and (2) intermolecular crosslinked peptides. Information about intramolecular crosslinking was obtained from reaction products in which both functional groups of the crosslinker reacted with lysine residues that reside in the same polypeptide chain (see Figures 4 and 5, Table 1). These peptides were sequenced by MS/MS and corresponded to the intramolecular crosslinking of: (1) residues K88 and K95 in (TLK88NITETCK95ACAQVNASKS), (2) residues K31 and K33 in LGAYDK31TK33K, (3) residues K95 and K104 in NITETCK95ACAQVNASK104S, (4) and residues K88 and K104 in TLK88NITETCKACAQVNASK104S. The informative contact position of lysines identified with the looplinks (see Table 1), was used to analyze the 3D crystal structure of the NTD of Mo-MLV integrase (3NNQ) in PDB data bank. We used this structure as a template and examined the distances between the crosslinked lysines. A maximum limit for productive crosslinking of 21.3 Å has been used as a restraint for the distance between the Cβ of two crosslinked lysines using BS3[22]. The distances between the crosslinked lysines were within the 21.3 Å limit, although the crosslinked K88-K104 was near this limit. Thus, our results are in agreement with the monomeric structure of the NTD domain in 3NNQ (Table 1).


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)

LC-MS/MS peptide map profile of N-terminal domain of Mo-MLV integrase monomer crosslinked gel band digested with trypsin. (A), in the sample the peak corresponds to the peptide 86–105 with two cross-linked lysines (K88 and K95). (B), the peak corresponds at the peptide 25–34 with intramolecular cross-linked between K31 and K33, (C) in the monomeric sample the peak corresponds at the peptide 89–105 with intramolecular cross-linked between K95 and K104.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: LC-MS/MS peptide map profile of N-terminal domain of Mo-MLV integrase monomer crosslinked gel band digested with trypsin. (A), in the sample the peak corresponds to the peptide 86–105 with two cross-linked lysines (K88 and K95). (B), the peak corresponds at the peptide 25–34 with intramolecular cross-linked between K31 and K33, (C) in the monomeric sample the peak corresponds at the peptide 89–105 with intramolecular cross-linked between K95 and K104.
Mentions: Chemical crosslinking combined with mass spectrometry (MS) provides a useful method for inferring sites of protein-protein interactions and for mapping the topology of protein complexes. We used the bi-functional crosslinking reagent BS3 that reacts with primary amine groups of lysines and the NTD integrase of Mo-MLV. In these experiments IN 1–105 was crosslinked with BS3 and the dimeric and monomeric band was separated by SDS-PAGE, subjected to digestion with trypsin or chymotrypsin and analyzed by LC-MS/MS as indicated in Materials and Methods, being compared with the MS/MS profile of the unmodified IN 1–105, sequencing only differential peaks (Tables 1 and 2). In these cases only two informative sequences were used and classified as (1) intramolecular crosslinked peptides (or looplinks) and (2) intermolecular crosslinked peptides. Information about intramolecular crosslinking was obtained from reaction products in which both functional groups of the crosslinker reacted with lysine residues that reside in the same polypeptide chain (see Figures 4 and 5, Table 1). These peptides were sequenced by MS/MS and corresponded to the intramolecular crosslinking of: (1) residues K88 and K95 in (TLK88NITETCK95ACAQVNASKS), (2) residues K31 and K33 in LGAYDK31TK33K, (3) residues K95 and K104 in NITETCK95ACAQVNASK104S, (4) and residues K88 and K104 in TLK88NITETCKACAQVNASK104S. The informative contact position of lysines identified with the looplinks (see Table 1), was used to analyze the 3D crystal structure of the NTD of Mo-MLV integrase (3NNQ) in PDB data bank. We used this structure as a template and examined the distances between the crosslinked lysines. A maximum limit for productive crosslinking of 21.3 Å has been used as a restraint for the distance between the Cβ of two crosslinked lysines using BS3[22]. The distances between the crosslinked lysines were within the 21.3 Å limit, although the crosslinked K88-K104 was near this limit. Thus, our results are in agreement with the monomeric structure of the NTD domain in 3NNQ (Table 1).

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