Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

Abstract

BackgroundWe 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.Methodology/ResultsPhotoaffinity 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.ConclusionAnalysis 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.

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

“…To circumvent the problem of low sequence specificity in forming a uniform IN-DNA complex, we employed protein-DNA cross-linking [14], [28]. A thiol group was attached to the 3′ OH end of a pre-cleaved viral gain-of-function (G) U3 DNA substrate [29] and was cross-linked to a cysteine residue introduced in/near the active site of IN through a disulfide bond.…”

“…PFV IN shares only a ∼15% sequence identity with HIV IN and is ∼100 amino acids (aa) larger than HIV IN, comprising an additional NTD extension domain (48 aa) and longer inter-domain linkers. Therefore, structural features of functional IN-DNA complexes distal to the active site may not be strictly conserved between PFV IN and the smaller three-domain IN including HIV and RSV [14]. RSV IN shares ∼25% sequence identity with HIV IN and the two proteins are very similar to each other in size (286 vs. 288 aa, respectively) and the lengths of inter-domain linkers [3].…”

Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

“…In addition, the linkers between the N-terminal and catalytic core domains and in particular between the catalytic core and C-terminal domains are significantly longer in the distantly related four-domain PFV IN (6,7) than the corresponding linkers in RSV or HIV IN. Structure-based sequence alignments of full-length RSV, HIV, and PFV INs provide an overview of these important structural features (8).…”

C-Terminal Domain of Integrase Binds between the Two Active Sites

“…With a unique orientation for the ASV CTD, vDNA-contacting residues would not correspond to the same structural positions on the HIV and PFV INs. Therefore, experimental data implicating a specific ASV CTD residue in vDNA binding 36 may not be directly transferable to the HIV and PFV CTDs.…”

“…The crystallographic positions of the PFV L1 and L2 linkers lie between the CTD and the bound vDNA, blocking CTD/vDNA interactions found by cross-linking studies on HIV-IN and ASV-IN. 36 The PFV-IN 30 and HIV-IN 5,15,16 structures show similar contacts between the CCD and the NTD, but these two domains are connected by the L1 linker in very different ways (Figure 1B). The position of the CTD relative to the CCD 5,19 (Figure 1C) and the contacts of the CTD 17,18,30 vary greatly among the crystallographic structures of PFV, HIV, and ASV INs.…”