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
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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