2 research outputs found
Insights into the Mechanism by Which Interferon‑γ Basic Amino Acid Clusters Mediate Protein Binding to Heparan Sulfate
The extensive functional repertoire
of heparin and heparan sulfate,
which relies on their ability to interact with a large number of proteins,
has recently emerged. To understand the forces that drive such interactions
the binding of heparin to interferon-γ (IFNγ), used as
a model system, was investigated. NMR-based titration experiments
demonstrated the involvement of two adjacent cationic domains (D1:
KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal
sequence of the cytokine. Kinetic analysis showed that these two domains
contribute differently to the interaction: D1 is required to form
a complex and constitutes the actual binding site, whereas D2, although
unable to associate with heparin by itself, increased the association
rate of the binding. These data are consistent with the view that
D2, through nonspecific electrostatic forces, places the two molecules
in favorable orientations for productive binding within the encounter
complex. This mechanism was supported by electrostatic potential analysis
and thermodynamic investigations. They showed that D1 association
to heparin is driven by both favorable enthalpic and entropic contributions,
as expected for a binding sequence, but that D2 gives rise to entropic
penalty, which opposes binding in a thermodynamic sense. The binding
mechanism described herein, by which the D2 domain kinetically drives
the interaction, has important functional consequences and gives a
structural framework to better understand how specific are the interactions
between proteins and heparin
Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A
Hepatitis
C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding
phosphoprotein composed of a N-terminal membrane anchor (AH), a structured
domain 1 (D1), and two intrinsically disordered domains (D2 and D3).
The knowledge of the functional architecture of this multifunctional
protein remains limited. We report here that NS5A-D1D2D3 produced
in a wheat germ cell-free system is obtained under a highly phosphorylated
state. Its NMR analysis revealed that these phosphorylations do not
change the disordered nature of D2 and D3 domains but increase the
number of conformers due to partial phosphorylations. By combining
NMR and small angle X-ray scattering, we performed a comparative structural
characterization of unphosphorylated recombinant D2 domains of JFH1
(genotype 2a) and the Con1 (genotype 1b) strains produced in <i>Escherichia coli</i>. These analyses highlighted a higher intrinsic
folding of the latter, revealing the variability of intrinsic conformations
in HCV genotypes. We also investigated the effect of D2 mutations
conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors
on the structure of the recombinant D2 Con1 mutants and their binding
to CypA. Although resistance mutations D320E and R318W could induce
some local and/or global folding perturbation, which could thus affect
the kinetics of conformer interconversions, they do not significantly
affect the kinetics of CypA/D2 interaction measured by surface plasmon
resonance (SPR). The combination of all our data led us to build a
model of the overall structure of NS5A, which provides a useful template
for further investigations of the structural and functional features
of this enigmatic protein