Structural study of LEDGF cellular binding partners

 

K. Prochazkova 1, M. Horejsi 2, K. Cermakova 1, P. Rezacova 1,2.

 

1Institute of Organic Chemistry and BiochemistryAS CR, Flemingovo nam. 2, 166 10, Prague 6, Czech Republic

2Insitute of Molecular Genetics AS CR, Flemingovo nam. 2, 166 10, Prague 6, Czech Republic

katerina.prochazkova@uochb.cas.cz

rezacova@img.cas.cz

 

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a prominent interaction partner of human immunodeficiency virus type 1 (HIV) integrase and co-factor of 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). Structural information on interaction between HIV integrase and LEDGF/p75 become 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 IBD, interaction domain of LEDGF/p75 was cloned and expressed in E. coli. The protein was isolated from inclusion bodies and purified with yields sufficient for binding studies. The JPO2 was cloned as a full-length protein, expressed in E. coli and optimization of purification procedure is in progress now. From pogZ, the DDE domain responsible for interaction with LEDGF/p75 was cloned, expressed in E. coli and purified from the cytosol. The DDE domain of pogZ (residues 1117-1323) is a putative catalytic domain comprising Asp and Glu residues in the catalytic site. Pre-crystallization analyses and crystallization trials were initiated.

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.

References

1.     P. Cherepanov, A. L. Ambrosio, S. Rahman, T. Ellenberger, A. Engelman, Proc. Natl. Acad. Sci. U.S.A., 102, (2005), 17308.

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.

 

Acknowledgements

This work was supported by grant from the FP7 framework of the European Union (THINC, HEALTH-2007-2.3.2-1) and in part by projects nos. AV0Z40550506 and AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic.