Structure of hemachatoxin.

<p>(<b>A</b>) Ribbon representation of the hemachatoxin monomer. Cysteine bonds are shown in <i>yellow.</i> β-strands, N- and C- terminals are labeled. (<b>B</b>) Electron density map<b>.</b> A sample final <i>2Fo-Fc</i> map of hemachatoxin shows the region from Tyr23 to Lys29. The map is contoured at a level of 1σ. (<b>C</b>) The electrostatic surface potential of hemachatoxin is shown in the same orientation as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone-0048112-g003" target="_blank">Figure 3A</a>. Blue indicates positive potential and red indicates negative potential in units kT/e. All the structure related figures of this paper were prepared using the program PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Delano1" target="_blank">[77]</a>.</p

Multiple sequence alignment of hemachatoxin with cardiotoxins/cytotoxins (A) and other three-finger toxins (B).

<p>Toxin names, species and accession numbers are shown. Conserved residues in all the sequences are highlighted in black. The type of cardiotoxin based on the conserved Pro31 is highlighted in grey. Disulfide linkages and loop regions are also shown. The sequence identity (in percentage) of each protein with hemachatoxin is shown at the end of each sequence.</p

Purification of hemachatoxin from the venom of <i>H. haemachatus</i>.

<p>(<b>A</b>) Size-exclusion chromatogram of the crude venom. The proteins were eluted using 50 mM Tris-HCl, pH 7.4 and monitored at 280 nm. The fractions of peak 3 (<i>black horizontal bar</i>) were pooled and sub-fractionated on RP-HPLC. (<b>B</b>) RP-HPLC chromatogram of peak 3 using a linear gradient of 28–50% solvent B. The elution was monitored at 215 nm. The <i>black arrow</i> indicates the elution of hemachatoxin. (<b>C</b>) The re-purification of hemachatoxin on a shallow gradient of 35–45% solvent B. The elution was monitored at 215 nm. (<b>D</b>) The ESI-MS profile of hemachatoxin showing the three peaks of mass/charge (m/z) ratio ranging from +4 to +6 charges. The mass of hemachatoxin was determined to be 6835.68±0.94 Da.</p

Crystallographic data and refinement statistics.

<p>Statistics from the current model.</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>Values in the parenthesis are the highest resolution bin values.</p></i></i

Comparison of hemachatoxin with other three-finger toxins.

<p>(<b>A</b>) Structure based sequence alignment of hemachatoxin and its homologs, cardiotoxin 3 (1H0J), cytotoxin 3 (1XT3), cardiotoxin A3 (2BHI), cardiotoxin VI (1UG4) and cardiotoxin V (1KXI), (all from <i>Naja atra</i>), cardiotoxin V_II4 (1CDT) from <i>Naja mossambica</i> and toxin-γ (1TGX) (a cardiotoxin from <i>Naja nigricollis</i>). This figure was generated using the programs ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Larkin1" target="_blank">[78]</a> and ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Gouet1" target="_blank">[79]</a>. (<b>B</b>) Comparison of hemachatoxin with its structural homologs. Hemachatoxin (brown), cardiotoxin 3 [1H0J] (cyan), cytotoxin 3 [1XT3] (black), carditotoxin A3 [2BHI] (blue), cardiotoxin VI [1UG4] (red), cardiototoxin V [1KXI] (pink), cardiotoxin V_II4 [1CDT] (green) and toxin-γ [1TGX] (yellow).</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

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

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

Substitution of key residues disrupts dimerization.

<p>(A) Analytical ultra-centrifugation profile of HEPN Asn186Asp domain of VBP. HEPN domain with Asn186Asp substitution sediments as a single species at an apparent molecular mass of 18.5 kDa. (B) Analytical ultra-centrifugation profile of VBP Asn186Asp. VBP with Asn186Asp substitution sediments as a single species at an apparent molecular mass of 37.5 kDa. Abbreviations: HEPN, higher eukaryotes and prokaryotes nucleotide (domain).</p

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