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Directed Evolution of a Beta-Sheet Scaffold for Targeting Proteins Involved in Human Disease - Thrombin and the Vascular Endothelial Growth Factor (VEGF)
Aberrant protein-protein interactions have been implicated in numerous human diseases. Hence it becomes important to understand the physicochemical basis of these interactions as well as to develop methods to selectively target these protein surfaces. However, it is difficult to target such protein surfaces by small molecules as these surfaces (≥ 600 Ų) are large and flat. We demonstrate the feasibility of utilizing a small beta-sheet scaffold for targeting thrombin as proof of principle. Thrombin is a trypsinlike serine protease generated in the penultimate step of the blood coagulation cascade. Thrombin has numerous potential interaction sites to test our methodology. This strategy was further extended to target the Vascular Endothelial Growth Factor (VEGF). VEGF is a disulfide-linked cytokine that exerts its activity by binding to two high affinity receptors. VEGF has been implicated in angiogenesis where the growth of new blood capillaries provides nourishment to tumor cells and damage delicate retinal tissues. This will help us to develop a new scaffold and provide essential chemical and structural information necessary for binding these discrete protein surfaces. Furthermore, this facilitates the subsequent transfer of minimal epitope information to a small molecule
A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins Utilizing Split-Luciferase Reassembly
The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.
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Covalent Conjugation of a Peptide Triazole to HIV‑1 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 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
Conformational and structural features of HIV-1 gp120 underlying the dual receptor antagonism by cross-reactive neutralizing antibody m18
We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1 YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (K D ≈ 5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.(Figure Presented) © 2011 American Chemical Society.link_to_subscribed_fulltex