Abstract
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.
Highlights
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 catalytic core domain (CCD) and C-terminal domain (CTD) [12] or N-terminal domain (NTD) and CCD [13] likewise unveiled dimeric organizations
RNA interference (RNAi)-mediated knock-down of endogenous lens epithelium-derived growth factor (LEDGF) to below detectable levels resulted in reduction of infection to 3.5% of that observed in the presence of normal cells [33]
We previously reported that LEDGF significantly stimulated the in vitro activities of HIV-1 IN whereas the isolated IN-binding domain (IBD) failed to do so [39, 40]
Summary
Expression Plasmids and Recombinant Proteins—HIV-1 IN proteins were expressed from pKBIN6Hthr, which was derived from pKB-IN6H [28] by replacing amino acids VDKLAAALE upstream from the C-terminal His affinity tag with LVPRGSALE (thrombin cleavage site underlined) by PCR-directed mutagenesis. Mass Spectrometric Footprinting—In parallel reactions, free IN and INϩLEDGF were first incubated at room temperature for 30 min and subjected to treatments at 37 °C with 1 mM N-hydroxysuccinimide (NHS)-biotin for 30 min or 20 mM p-hydroxyphenylglyoxal (HPG) for 60 min These concentrations of modifying reagents were chosen because comparative pulldown experiments with untreated and modified IN-LEDGF complexes indicated that under these conditions the integrity of the preassembled protein-protein complex was fully preserved (data not shown). LEDGF Binding Affinities to Wild-type and Mutant INs— LEDGF (50 – 650 nM) was incubated with 100 nM His-tagged IN (WT or mutant) in binding buffer (50 mM Hepes (pH 7.1), 200 mM NaCl, 2 mM MgCl2, 100 mM imidazole, 0.1% (v/v) Nonidet P40) for 60 min at room temperature. Nonspecific signal was not detected when LEDGF was incubated with Ni-NTA beads in the absence of IN (data not shown)
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