Dithiothreitol Causes HIV-1 Integrase Dimer Dissociation While Agents Interacting With the Integrase Dimer Interface Promote Dimer Formation
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- 作者：Manuel Tsiang ; Gregg S. Jones ; Magdeleine Hung ; Dharmaraj Samuel ; Nikolai Novikov ; Susmith Mukund ; Katherine M. Brendza ; Anita Niedziela-Majka ; Debi Jin ; Xiaohong Liu ; Michael Mitchell ; Roman Sakowicz ; Romas Geleziunas
- 刊名：Biochemistry
- 出版年：2011
- 出版时间：March 15, 2011
- 年：2011
- 卷：50
- 期：10
- 页码：1567-1581
- 全文大小：1378K
- 年卷期：v.50,no.10(March 15, 2011)
- ISSN：1520-4995
文摘
We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose鈭抮esponse curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose鈭抮esponse behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN鈭扡EDGF interaction also stabilize IN dimers and promote their formation.