Structural study of LEDGF/p75 binding partners

 

Petr Těšina1, Kateřina Čermáková1, Kateřina Procházková1, Magdaléna Hořejší2,

Frauke Christ3, Jan De Rijck3, Václav Veverka1 and Pavlína Řezáčová1

 

1Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nam. 2, 166 10, Prague, Czech Republic

2Institute of Molecular Genetics AS CR, Flemingovo nam. 2, 166 10, Prague, Czech Republic

3Molecular Medicine, KULeuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium

tesina@uochb.cas.cz

 

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a prominent cellular cofactor for human immunodeficiency virus (HIV) integration. LEDGF/p75 tethers the preintegration complex to the host chromosome and this process is crucial for HIV replication. HIV integrase interacts with the C-terminal part of LEDGF/p75, region designated integrase-binding domain (IBD, amino acids residues 347 - 429). Interaction interface between HIV integrase and LEDGF/p75 became an attractive target for design of small molecule inhibitors blocking this interaction [1].

While the role of LEDGF/p75 in HIV integration is well characterized, very little is known about its physiological function. As a transcriptional co-activator, LEDGF/p75 is implicated not only in HIV replication, but also in human cancer and autoimmunity. The LEDGF/p75 was shown to interact through its IBD with several cellular proteins and recent evidence implies that LEDGF/p75 is a general adaptor protein tethering various factors to chromatin [2].

In this work, we set to prepare two LEDGF/p75 physiological binding partners JPO2 [2] and pogo transposable element (pogZ) [3]. The aim of our study is to obtain structural information on the LEDGF/p75 interaction with its physiological binding partners JPO2 and pogZ, respectively. Such structural information is essential for understanding the LEDGF/p75 biological role and might help in design of inhibitors selectively blocking interaction with HIV integrase while not interfering with the LEDGF/p75 biological function.

The IBD, interaction domain of LEDGF/p75 was cloned and expressed in E. coli. The protein was purified with yields sufficient for binding and structural studies. The JPO2 and pogZ were cloned and isolated as a full-length proteins and several alternative constructs encompassing individual domains with IBD binding affinity. Both proteins were biophysically characterized as well as the reconstituted complex of JPO2 and IBD. The JPO2 protein was found to be intrinsically flexible, which prevented us getting any crystals and forced us into an alternative path of structural characterization of the complex of JPO2 and IBD.

This work was supported by grants from the FP7 framework of the European Union (THINC, HEALTH-2007-2.3.2-1) and the programme ‘Navrat‘ MSMT (LK11205)

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2.     G. N. Maertens, P. Cherepanov, A. Engelman, J Cell Sci, 119, (2006), 2563.

3.     K. Bartholomeeusen, F. Christ, J. Hendrix, J. C.  Rain, S. Emiliani, R. Benarous, Z.  Debyser, R. Gijsbers, J. De Rijck, J Biol Chem.,  284, (2009), 11467.