3 research outputs found
Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer
We recently reported the discovery
of a recombinant chimera, denoted
DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively
cause specific and potent lytic inactivation of both pseudotyped and
fully infectious human immunodeficiency virus (HIV-1) virions. The
chimera is composed of the lectin cyanovirin-N (CVN) fused to the
20-residue membrane-proximal external region (MPER) of HIV-1 gp41.
Because the Env gp120-binding CVN domain on its own is not lytic,
we sought here to determine how the MPER<sub>(DAVEI)</sub> domain
is able to endow the chimera with virolytic activity. We used a protein
engineering strategy to identify molecular determinants of MPER<sub>(DAVEI)</sub> that are important for function. Recombinant mutagenesis
and truncation demonstrated that the MPER<sub>(DAVEI)</sub> domain
could be significantly minimized without loss of function. The dependence
of lysis on specific MPER sequences of DAVEI, determination of minimal
linker length, and competition by a simplified MPER surrogate peptide
suggested that the MPER domain of DAVEI interacts with the Env spike
trimer, likely with the gp41 region. This conclusion was further supported
by observations from binding of the biotinylated MPER surrogate peptide
to Env protein expressed on cells, monoclonal antibody competition,
a direct binding enzyme-linked immunosorbent assay on viruses with
varying numbers of trimeric spikes on their surfaces, and comparison
of maximal interdomain spacing in DAVEI to that in high-resolution
structures of Env. The finding that MPER<sub>(DAVEI)</sub> in CVNâMPER
linker sequences can be minimized without loss of virolytic function
provides an improved experimental path for constructing size-minimized
DAVEI chimeras and molecular tools for determining how simultaneous
engagement of gp120 and gp41 by these chimeras can disrupt the metastable
virus Env spike
A Model of Peptide Triazole Entry Inhibitor Binding to HIVâ1 gp120 and the Mechanism of Bridging Sheet Disruption
Peptide
triazole (PT) entry inhibitors prevent HIV-1 infection by blocking
the binding of viral gp120 to both the HIV-1 receptor and the coreceptor
on target cells. Here, we used all-atom explicit solvent molecular
dynamics (MD) to propose a model for the encounter complex of the
peptide triazoles with gp120. Saturation transfer difference nuclear
magnetic resonance (STD NMR) and single-site mutagenesis experiments
were performed to test the simulation results. We found that docking
of the peptide to a conserved patch of residues lining the âF43
pocketâ of gp120 in a bridging sheet naiÌve gp120 conformation
of the glycoprotein led to a stable complex. This pose prevents formation
of the bridging sheet minidomain, which is required for receptorâcoreceptor
binding, providing a mechanistic basis for dual-site antagonism of
this class of inhibitors. Burial of the peptide triazole at the gp120
inner domainâouter domain interface significantly contributed
to complex stability and rationalizes the significant contribution
of hydrophobic triazole groups to peptide potency. Both the simulation
model and STD NMR experiments suggest that the I-X-W [where X is (2<i>S</i>,4<i>S</i>)-4-(4-phenyl-1<i>H</i>-1,2,3-triazol-1-yl)Âpyrrolidine]
tripartite hydrophobic motif in the peptide is the major contributor
of contacts at the gp120âPT interface. Because the model predicts
that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes
to the stability of the PTâgp120 complex, we tested this prediction
through analysis of peptide binding to gp120 mutant S375A. The results
showed that a peptide triazole KR21 inhibits S375A with 20-fold less
potency than WT, consistent with predictions of the model. Overall,
the PTâgp120 model provides a starting point for both the rational
design of higher-affinity peptide triazoles and the development of
structure-minimized entry inhibitors that can trap gp120 into an inactive
conformation and prevent infection
Covalent Conjugation of a Peptide Triazole to HIVâ1 gp120 Enables Intramolecular Binding Site Occupancy
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 hostâcell 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 PTâgp120 complex as a
blueprint to design a covalently conjugated PTâgp120 recombinant
protein. Initially, a single-cysteine gp120 mutant, E275C<sub>YUâ2</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 AE21âE275C
covalent conjugate. Surface plasmon resonance interaction analysis
revealed that the PTâgp120 conjugate exhibited suppressed binding
of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope
protein. Similar to the noncovalent PTâgp120 complex, the covalent
conjugate was able to bind the conformationally dependent mAb 2G12.
The results argue that the PTâgp120 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 PTâgp120 conjugate to the noncovalent
PTâgp120 complex support the orientation of binding of PT to
gp120 predicted in the molecular dynamics simulation model of the
PTâgp120 noncovalent complex. The conformationally stabilized
covalent conjugate can be used to expand the structural definition
of the PT-induced âoffâ 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