Assembly and budding in the late-stage of human immunodeficiency virus
type 1 (HIV-1) production relies on the polymerization of Gag protein at
the inner leaflet of the plasma membrane. We previously generated an
ankyrin repeat protein (Ank1D4) that specifically interacts with the
CAp24 protein. This study aimed to improve the binding activity of
Ank1D4 by generating two platforms for the Ank1D4 dimer. The design of
these constructs featured a distinct orientation of monomeric Ank1D4
connected by a linker peptide (G 4S) 4.
The binding surfaces in either dimer generated from the C-terminus of
the Ank1D4 monomer linked with the N-terminus of another monomer (Ank1D4
NC-NC) or its inverted form (Ank1D4
NC-CN), similar to monomeric Ank1D4. The interaction of
Ank1D4 NC-CN with CAp24 from capture ELISA was
significantly greater than that of Ank1D4 NC-NC and the
parental Ank1D4. The bifunctional characteristic of Ank1D4
NC-CN was further demonstrated using sandwich ELISA. The
binding kinetics of these ankyrins were evaluated using bio-layer
interferometry analysis. The K D of Ank1D4
NC-CN, Ank1D4 NC-NC and monomeric Ank1D4
was 3.5 nM, 53.7 nM, and 126.2 nM, respectively. The dynamics of the
interdomain linker and the behavior of ankyrin dimers were investigated
in silico. Upon the binding distance calculation from the
candidate structures, the achievement in obtaining double active sites
is more possible in Ank1D4 NC-CN. The CD spectroscopic
data indicated that secondary structure of dimer forms resemble Ank1D4
monomer α-helical content. This finding confers the strategy to generate
dimer from rigid scaffold for acquiring the binding avidity.