Abstract
BackgroundRetroviral 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Retroviral integrases (INs) catalyze the integration of viral DNA in the chromosomal DNA of the infected cell
There is no information on the structure of the dimer of Mo-MLV in solution and how it interacts with the other IN domains
All retroviral integrases contain a HHCC zinc finger motif at the N-terminal domain that is involved in protein-protein interactions, which is essential for retroviral integration
Summary
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. 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. Protein-protein interactions play a fundamental role on the assembly of multimeric complexes of IN to carry out two-end concerted DNA integration [1,2,3]. The X-ray structure of a tetrameric integrase complex of the PFV IN and processed U5 DNA was solved [14,15,16]. The interaction between the NTDs in the latter dimer was detected by chemical crosslinking [18]
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