The integrase protein (IN) of HIV-1 catalyses the integration of the viral genome into the host chromosome. IN binds the viral DNA as a high-order oligomer. The cellular protein LEDGF/p75 binds IN and stimulates its activity. Our research aim is: (1) To reveal the active oligomerization state of IN, and how LEDGF/p75 affects it (2) To characterize IN binding to the viral DNA and to LEDGF/p75 (3) To reveal what is the mechanism in which LEDGF/p75 enhances IN activity. Based on the IN-LEDGF/p75 crystal structure complex, we have synthesized a set of fluorecein-labeled peptides derived from the IN binding loops of LEDGF/p75 and tested their IN binding using fluorescence anisotropy. IN bound the LEDGF361-370 peptides with Kd=3microM. Binding was highly cooperative, with Hill coefficient around 4, indicating tetramerization of IN upon peptide binding. IN did not bind a peptide from the second LEDGF loop. Binding of IN to 36-bp viral LTR DNA was cooperative with a Kd=37nM, and, Hill coefficient of 2, indicating binding of IN dimer to the DNA. A truncated IN mutant, IN52-288, did not bind the LEDGF peptides or the DNA, indicating involvement of IN1-51 in the binding process. LEDGF peptides did not alter the affinity of IN to DNA. Analytical gel filtration showed that free IN is highly aggregated, and binding to the LEDGF peptides or to DNA forces it out of the aggregate into its active dimeric or tetrameric form. Our results shed light on the molecular mechanism of IN-LEDGF/p75 binding.
|Original language||American English|
|Number of pages||1|
|Journal||Journal of Peptide Science|
|State||Published - 2006|