The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra-and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.Integration of the reverse-transcribed RNA genome into a host chromosome is an obligatory step for HIV-1 3 replication (reviewed in Ref. 1). This process is catalyzed by the retroviral enzyme integrase (IN) in two reaction steps. In the first step, which is called 3Ј-processing and takes place shortly after the cDNA is made, IN hydrolyzes a GT dinucleotide from each end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. The sites of attack on the two target DNA strands are separated by 5 bp, which leads to dissociation of the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. HIV-1 IN consists of three distinct structural and functional domains. The N-terminal domain (NTD) (residues 1-50) contains conserved pairs of histidine and cysteine residues that bind zinc (2, 3), which contributes to IN multimerization and its catalytic function (4, 5). The catalytic core domain (CCD) (residues 51-212) contains three acidic residues, Asp-64, Asp-116, and Glu-152, which play a key role in coordinating active site divalent metal ions (6, 7). The C-terminal domain (CTD) (residues 213-288) also contributes to functional IN multimerization (8, 9). Results of structural biology studies revealed each individual domain as a dimer (3,6,7,10,11) and more recent two-domain crystal structures comprised of the CCD and CTD (12) or NTD and CCD (13)...
Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.
To identify functional contacts between HIV-1 integrase (IN)and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys-and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The footprinting studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes. HIV-1 integrase (IN)4 is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for viral replication, there is no closely related cellular equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting reverse transcriptase (RT), protease, and virus-cell fusion. Detailed structural information on functional IN-DNA complexes could aid drug design efforts. For example, the promising diketo acid class of inhibitors preferentially bind to the assembled IN-viral DNA complex rather than the free protein (1-4).The two chemical reactions catalyzed by HIV-1 IN, 3Ј processing and DNA strand transfer, have been characterized in detail (reviewed in Ref. 5). First, IN removes two nucleotides from each 3Ј-end of the viral DNA synthesized by reverse transcriptase. In the following step, concerted transesterification reactions covalently join the viral DNA ends into the host genome (6). In vivo, the enzyme acts in the context of a large nucleoprotein complex with a number of viral and host proteins contributing to the integration process (7-21).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (NTD) (residues 1-50) that contains an HHCC zinc binding motif, the catalytic core domain (CCD) (residues 51-212) containing the DDE motif essential for coordinating catalytic divalent metals, and the C-terminal domain (CTD) (residues 213-288) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (22-26). In addition, two-domain CCD/ CTD (27) and NTD/CCD (28) crystal structures have been determined. However,...
HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the smallmolecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.
Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review.
We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein-protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein-protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein-protein interactions that are less amenable for crystallographic or NMR analysis.
Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein-protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein-peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a pre-defined raster of MALDI-TOF MS spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium-tin oxide. Further, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI-TOF instruments and has general utility for the label-free analysis of microarray assays.
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