Molecular mechanism of LEDGF/p75 dimerization
Vanda Lux 1, Tine Brouns 2, Kateřina
Čermáková 1,3, Pavel Srb 1, Milan Fábry 4,
Marcela Mádlíková 1, Magdalena Hořejší 4, Petr Novák 5,
Michael Kugler 1, Jiří Brynda 1,4, Zeger Debyser 2,
and Václav Veverka 1
1Institute of Organic
Chemistry and Biochemistry of the CAS, Prague, Czech Republic
2KU Leuven, Molecular
Virology and Gene Therapy, Leuven, Flanders, Belgium
3Department of
Molecular & Cellular Biology, Center for Precision Environmental Health,
and Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine,
Houston, TX, USA
4Institute of Molecular
Genetics of the CAS, Prague, Czech Republic
5Institute of Microbiology of the CAS, Prague, Czech
Republic
6Department of Cell
Biology, Faculty of Science, Charles University, Prague, Czech
Protein dimerization of many
eukaryotic transcription regulatory factors is critical for regulation of their
functional complexes. Recently it was shown, that transcription regulatory role
of an epigenetic reader Lens Epithelium Derived Growth Factor/p75 (LEDGF/p75,
also known as PSIP1) requires at least two copies of this protein to overcome
the nucleosome-induced barrier to transcription elongation. Moreover, various
LEDGF/p75 binding partners are enriched for dimeric features further
underscoring functional regulatory role of LEDGF/p75 dimerization. Here, we
used a combination of biophysical and biochemical techniques to investigate the
mechanism of LEDGF/p75 dimerization and its effect on molecular interactions
with other proteins. We dissected the minimal dimerization region in the
C-terminal part of LEDGF/p75 and with the help of paramagnetic NMR spectroscopy
identified the key molecular contacts that were used to refine the solution
structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by
domain-swapping within the integrase binding domain and additional
electrostatic ‘stapling’ of the negatively charged a-helix formed in the intrinsically disordered
C-terminal region. We validated mechanism of dimer formation using
structure-inspired dimerization defective LEDGF/p75 variants and chemical
cross-linking coupled to mass spectrometry. We also show how dimerization might
impact the LEDGF/p75 interactome.
This work was supported by grants
from the GACR (grant No. 16-06357S to V.V )