title: Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis.
abstract: BACKGROUND
  We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.
  
  
  RESULTS
  Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.
  
  
  CONCLUSIONS
  Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.
authors.affiliation: Biolinx LLC, Alburgh, Vermont, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Peletskaya
authors.forename: Mark
authors.initials: M
authors.lastname: Andrake
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.forename: George
authors.initials: G
authors.lastname: Merkel
authors.forename: Jerry
authors.initials: J
authors.lastname: Alexandratos
authors.forename: Dongwen
authors.initials: D
authors.lastname: Zhou
authors.forename: Ravi S
authors.initials: RS
authors.lastname: Bojja
authors.forename: Tadashi
authors.initials: T
authors.lastname: Satoh
authors.forename: Mikhail
authors.initials: M
authors.lastname: Potapov
authors.forename: Alex
authors.initials: A
authors.lastname: Kogon
authors.forename: Viktor
authors.initials: V
authors.lastname: Potapov
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Anna Marie
authors.initials: AM
authors.lastname: Skalka
mesh_terms: D000595:Amino Acid Sequence; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003432:Cross-Linking Reagents; D004279:DNA, Viral; D019426:Integrases