2 research outputs found
Comparative Thermodynamic Analysis of Zinc Binding to the His/Cys Motif in Virion Infectivity Factor
HIV-1
virion infectivity factor (Vif) is an accessory protein that induces
the proteasomal degradation of the host restriction factor, apolipoprotein
B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). Degradation
of APOBEC3G requires the interaction of Vif with Cul5, the scaffold
for an E3 ubiquitin ligase. A highly conserved region in HIV-1 Vif
termed the HCCH motif binds zinc and is critical for recruitment of
Cul5 and degradation of APOBEC3G. To gain thermodynamic and mechanistic
insight into zinc binding to diverse Vif proteins, we have employed
a combination of isothermal titration calorimetry, analytical ultracentrifugation,
and Cul5 pull down assays. The proton linkage of zinc binding to HIV-1
Vif was analyzed under different buffer conditions and consistent
with the release of two Cys-thiol protons upon zinc binding, supporting
earlier EXAFS studies. Zinc binding to Vif proteins from HIV-1, SIV<sub>Agm</sub>, HIV-2, and SIV<sub>Mac</sub> followed a trend in which
the enthalpy of zinc binding became less favorable and the entropy
of zinc binding became more favorable. Using AUC, we determined that
zinc induced oligomerization of Vif proteins from HIV-1 and SIV<sub>Agm</sub> but had little or no effect on the oligomeric properties
of Vif proteins from HIV-2 and SIV<sub>Mac</sub>. The zinc dependence
of Cul5 recruitment by Vif was investigated. All Vif proteins except
HIV-2 Vif required zinc to stabilize the interaction with Cul5. The
trends in enthalpy–entropy compensation, zinc-induced oligomerization,
and Cul5 recruitment are discussed in terms of the <i>apo</i> conformation of the HCCH motif and the role of zinc in stabilizing
the structure of Vif
Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding
The
ligand-induced
conformational changes of periplasmic binding
proteins (PBP) play a key role in the acquisition of metabolites in
ATP binding cassette (ABC) transport systems. This conformational
change allows for differential recognition of the ligand occupancy
of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis
in the transporter, a phenomenon in which ATP hydrolysis is not coupled
to metabolite transport. In many systems, the PBP conformational change
is insufficient at eliminating futile ATP hydrolysis. Here we identify
an additional state of the PBP that is also allosterically regulated
by the ligand. Ligand binding to the homodimeric apo PBP leads to
a tightening of the interface α-helices so that the hydrogen
bonding pattern shifts to that of a 3<sub>10</sub> helix, in-turn
altering the contacts and the dynamics of the protein interface so
that the monomer exists in the presence of ligand