Covalent Conjugation of a Peptide Triazole to HIV‑1
gp120 Enables Intramolecular Binding Site Occupancy
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Abstract
The HIV-1 gp120 glycoprotein is the
main viral surface protein
responsible for initiation of the entry process and, as such, can
be targeted for the development of entry inhibitors. We previously
identified a class of broadly active peptide triazole (PT) dual antagonists
that inhibit gp120 interactions at both its target receptor and coreceptor
binding sites, induce shedding of gp120 from virus particles prior
to host–cell encounter, and consequently can prevent viral
entry and infection. However, our understanding of the conformational
alterations in gp120 by which PT elicits its dual receptor antagonism
and virus inactivation functions is limited. Here, we used a recently
developed computational model of the PT–gp120 complex as a
blueprint to design a covalently conjugated PT–gp120 recombinant
protein. Initially, a single-cysteine gp120 mutant, E275C<sub>YU‑2</sub>, was expressed and characterized. This variant retains excellent
binding affinity for peptide triazoles, for sCD4 and other CD4 binding
site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds
the coreceptor recognition site. In parallel, we synthesized a PEGylated
and biotinylated peptide triazole variant that retained gp120 binding
activity. An N-terminally maleimido variant of this PEGylated PT,
denoted AE21, was conjugated to E275C gp120 to produce the AE21–E275C
covalent conjugate. Surface plasmon resonance interaction analysis
revealed that the PT–gp120 conjugate exhibited suppressed binding
of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope
protein. Similar to the noncovalent PT–gp120 complex, the covalent
conjugate was able to bind the conformationally dependent mAb 2G12.
The results argue that the PT–gp120 conjugate is structurally
organized, with an intramolecular interaction between the PT and gp120
domains, and that this structured state embodies a conformationally
entrapped gp120 with an altered bridging sheet but intact 2G12 epitope.
The similarities of the PT–gp120 conjugate to the noncovalent
PT–gp120 complex support the orientation of binding of PT to
gp120 predicted in the molecular dynamics simulation model of the
PT–gp120 noncovalent complex. The conformationally stabilized
covalent conjugate can be used to expand the structural definition
of the PT-induced “off” state of gp120, for example,
by high-resolution structural analysis. Such structures could provide
a guide for improving the subsequent structure-based design of inhibitors
with the peptide triazole mode of action