have applied small angle X-ray scattering (SAXS) and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain (NTD) intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the NTD of one monomer with the C-terminal domain (CTD) and catalytic core domain of the second monomer, as well as direct interactions between the two CTDs. This organization is similar to the "reaching dimer" previously described for wild type ASV apo-IN, and resembles the inner, substrate-binding dimer in the crystal structure of the PFV intasome. Results from our SAXS and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.
"The HIV-1 IN CCD (52–210) dimer interface buries 25% more area than FIV IN CCD (61–212; 1,500 A ˚ 2 versus 1,200 A ˚ 2 ) and involves twice as many hydrophobic contacts (99 versus 43), explaining the susceptibility of FIV IN, but not HIV-1, to monomerization by a single F187K mutation. It is well recognized that the tetrameric configuration of apo-IN is dissimilar to that of IN assembled upon DNA binding, and that IN must exist in lower-order oligomeric states in order to interact with the substrate DNA and form functional intasomes (Alian et al., 2009; Barsov et al., 1996; Bojja et al., 2013; Lesbats et al., 2008). Whereas the form of these smaller-multimeric IN entities , likely dimers, and their interaction interfaces and assembly pathways are yet to be defined, we suggest that the residue at the C-terminal tip of CCD a-5 (F187 in FIV) may pin the dimer interface, which hinges the two protomers together during flexible IN rearrangements upon DNA binding. "
[Show abstract][Hide abstract] ABSTRACT: Retroviral DNA integration into the host genome is mediated by nucleoprotein assemblies containing tetramers of viral integrase (IN). Whereas the fully active form of IN comprises a dimer of dimers, the molecular basis of IN multimerization has not been fully characterized. IN has consistently been crystallized in an analogous dimeric form in all crystallographic structures and experimental evidence as to the level of similarity between IN monomeric and dimeric conformations is missing because of the lack of IN monomeric structures. Here we identify Phe187 as a critical dimerization determinant of IN from feline immunodeficiency virus (FIV), a nonprimate lentivirus that causes AIDS in the natural host, and report, in addition to a canonical dimeric structure of the FIV IN core-domain, a monomeric structure revealing the preservation of the backbone structure between the two multimeric forms and suggest a role for Phe187 in "hinging" the flexible IN dimer.
"Differences in conformation reflect different combinations of interactions between the three domains of the enzyme. Crosslinking experiments in solution have revealed NTD-NTD interactions in the tetramer  that were not observed in the crystal structure of fragments of HIV-1 IN or PFV IN structure. It has been speculated that this kind of interaction may be related to a “domain swapping” phenomenon in which an interaction between the NTD and CCD domains is substituted by an interaction between the NTDs . "
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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 abstract][Hide abstract] ABSTRACT: Due to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3' processing reaction. The enzyme then uses the resulting viral DNA 3'-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5'-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3' processing, and the viral DNA 3'-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance.
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