Experimental screening for ligands binding to the Zn⁺²-free NS3 protease.

<p>Thermal denaturation curves of Zn⁺²-free NS3 protease followed by ANS fluorescence in the presence of different compounds (100 mM sodium acetate, pH 5, 2 mM EDTA). Inset: Thermal denaturation of Zn⁺²-free NS3 protease followed by tryptophan intrinsic fluorescence (average energy of spectra). The unfolding transition restricted to the temperature range accessible in the microplate fluorescence reader for the library screening is indicated in closed squares for comparison.</p

Dissociation constants for selected compounds binding to drug-resistance-associated NS3 protease variants (25°C, 100 mM sodium acetate, 2 mM EDTA, pH 5).

a<p>The inactive S139A mutant represents the pseudo-wild-type, behaving similarly to wild-type (WT) NS3 protease in terms of structural stability, substrate binding affinity and NS4A activation.</p><p>Relative error in the parameters is 15%.</p

Inhibition of HCV replicon in cell assays.

<p>Evaluation of potency and cytotoxicity of the selected compounds in cell assays. HCV replicon replication rate (white bars) and cell survival (closed squares) were assessed in cell culture at increasing compound concentration to determine EC50 and CC50. For compounds 1 and 3 assays with lower concentrations were performed in order to reliably determine the EC50. *UTC: untreated controls.</p

In vitro enzymatic inhibition of NS3 protease.

<p>(Left panel) Fluorescence intensity measured as a function of time (wavelengths of 380 nm and 500 nm for excitation and emission, respectively) for the substrate catalysis by NS3 protease in the absence (open squares) or presence of compound 1 at 25 µM (closed squares), in sodium acetate pH 5, 0.8 nM free Zn⁺² concentration. (Right panel) NS3 protease activity was determined as the initial slope of the curves. The percentage of activity is calculated as the quotient between the activity of NS3 protease in the presence (25 µM) and the absence of a given compound (compound 1, closed squares; compound 5, open squares).</p

Action mechanism of the allosteric inhibitors.

<p>The proper folding of the NS3 protease towards the active conformation, promoted <i>in vivo</i> by its two cofactors (Zn⁺² and NS4A), is prevented by the compounds through stabilization of the inactive partially folded Zn⁺²-free conformation.</p

Pull down assays.

<p>(A) MBP/MBP-VirD2 bound to amylose resin was incubated overnight with 6His-VBP, followed by washes. The final beads were resolved on a 12.5% SDS gel, transferred to a PVDF membrane and treated with anti-His monoclonal antibody (1∶10,000). 6His-VBP was loaded into the lane 5 as a reference. The VBP species used include: lane 1. Wild-type (WT) VBP; lane 2.VBP D173N; lane 3.VBP K184D; lane 4. VBP N186D; lane 5. VBP wild-type loaded on to the gel for reference; .lane 6. VBP passed through MBP bound to amylose beads. (B) 6His-VBP/substituted 6His-VBP bound to Ni-NTA metal affinity resin was incubated with freshly prepared <i>A. tumefaciens</i> crude extracts. After incubation at 4°C for 1 h, the resin was washed four times. The bound complex was eluted with 250 mM imidazole. The eluted protein was resolved on SDS-Gel, transferred to a PVDF membrane and the protein was detected using protein (VirD2 and VirD4 CP) specific monoclonal antibodies. The VBP species used include: lane 1. Crude extract loaded for reference; lane 2. WT VBP; lane 3.VBP D173N; lane 4.VBP K184D; lane 5. VBP N186D.</p

Crystallographic statistics and refinement details.

#<p>SAD – Single-wavelength anomalous diffraction.</p>a<p>R_sym = Σ |I_i−<i>|/Σ|I_i| where I_i is the intensity of the i^th measurement, and <i> is the mean intensity for that reflection.</i></i></p><i><i>b<p>R_work = Σ |F_obs−F_calc|/Σ|F_obs| where F_calc and F_obs are the calculated and observed structure factor amplitudes, respectively.</p>c<p>R_free = as for R_work, but for 10.0% of the total reflections chosen at random and omitted from refinement.</p><p>Individual B-factor refinement was carried out.</p><p>*Values in parentheses are for highest resolution bin.</p></i></i

The HEPN domain of VBP is the dimerization domain.

<p>(A) Analytical ultra-centrifugation profile of the HEPN domain of VBP. The HEPN domain sediments as a major species at an apparent molecular mass of 37 kDa. (B) Analytical ultra-centrifugation profile of the NT domain of VBP. The NT domain sediments as a major species at an apparent molecular mass of 17 kDa. Abbreviations: HEPN, higher eukaryotes and prokaryotes nucleotide binding domain; NT, Nucleotidyltransferase domain.</p

Schematic representation shows the induction of tumors in plants by <i>Agrobacterium tumefaciens</i> and the role of VBP.

<p>Our experiments show that only dimeric VBP can bind to VirD2 or VirD4 CP. We propose that the VirD2–T-DNA complex could possibly be recruited to the T4SS apparatus by two different mechanisms. Mechanism I: Dimeric VBP binds to VirD2 (steps 1 and 2), it recruits the VirD2–T-DNA complex to the (VirD4 CP) T4SS apparatus which constitutes the 11 VirB proteins (step 3). Mechanism II: Dimeric VBP binds to VirD4 CP (step 2). VBP acts as a docking station to recruit the VirD2–T-DNA complex (step 3). Once recruited to the T4SS apparatus, the VirD2–T-DNA complex is translocated into the host cell cytoplasm (step 4). It is yet unclear whether VBP is translocated or not <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003948#ppat.1003948-Guo1" target="_blank">[8]</a>. VirE2 is one among the several effector proteins that are translocated through the T4SS. Inside the host cell, VirE2 coats the single-stranded T-DNA to form the VirD2–T-DNA-VirE2 complex (step 5). Certain host cytoplasmic proteins recognize and bind to the nuclear localization signals on VirD2 and VirE2 and translocate the VirD2–T-DNA:VirE2 complex to the nucleus (step 6) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003948#ppat.1003948-Zupan1" target="_blank">[35]</a>. Inside the nucleus, VirD2 and VirE2, along with a plethora of host proteins, help the T-DNA to integrate with the host DNA (step 7) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003948#ppat.1003948-Citovsky2" target="_blank">[36]</a>. The integrated T-DNA modulates the host cell process to enable the bacterial colonization and growth, which leads to the formation of tumor (step 8). IM: Inner membrane; OM: Outer membrane; HCM: Host cell membrane; NM: Nuclear membrane.</p

VBP is a dimer.

<p>(A) Gel filtration profile of VBP. Full-length VBP elutes as a single peak (in green) at an elution volume corresponding to an apparent molecular mass of 75 kDa. The molecular weight standard is shown in red. The peak at 670 kDa corresponds to aggregated VBP that elutes in the void. (B) Analytical ultra-centrifugation profile of VBP. The full-length VBP sediments as a single species at an apparent molecular mass of 75 kDa.</p