Structural Determinants in Protein Folding: A Single Conserved Hydrophobic Residue Determines Folding of EGF Domains

The epidermal growth factor (EGF) domain is evolutionarily conserved despite hypervariability in amino acid sequences. They fold into a three-looped conformation with a disulfide pairing of C₁–C₃, C₂–C₄ ,and C₅–C₆. To elucidate the structural determinants that dictate the EGF fold, we selected the fourth and fifth EGF domains of thrombomodulin (TM) as models; the former domain folds into the canonical conformation, while the latter domain folds with alternate disulfide pairing of C₁–C₂, C₃–C₄, and C₅–C₆. Since their third disulfide (C₅–C₆) is conserved, we examined the folding tendencies of synthetic peptides corresponding to truncated domain four (t-TMEGF4) and five (t-TMEGF5), encompassing the segment C₁ to C₄. These peptides fold into their respective disulfide isoforms indicating that they contain all the required structural determinants. On the basis of the folding tendencies of these peptides in the absence and presence of 6 M Gn·HCl or 0.5 M NaCl, we determined that hydrophobic interactions are needed for the canonical EGF fold but not for the noncanonical fold. Sequence alignment of extant EGF domains and examination of their three-dimensional structures allowed us to identify a highly conserved hydrophobic residue in intercysteine loop 3 as the key contributor, which nucleates the hydrophobic core and acts as the lynch pin. When this hydrophobic residue (Tyr25) was substituted with a more hydrophilic Thr, the hydrophobic interactions were disrupted, and t-TMEGF4-Y25T folds similar to t-TMEGF5. Taken together, our results for the first time demonstrate that a single conserved hydrophobic residue acts as the key determinant in the folding of EGF domains

Applied cardiology : the journal of cardiovascular medicine

Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure–function relationships of Oh9-1, an Ω-neurotoxin from <i>Ophiophagus hannah</i> venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in <i>Xenopus</i> oocytes indicated Oh9-1 is selective for rat muscle type <i>α1β1εδ</i> (adult) and <i>α1β1γδ</i> (fetal) and rat neuronal <i>α3β2</i> subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to <i>α1β1εδ</i> and <i>α3β2</i> subtypes. Oh9-1’s loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at <i>α1β1εδ</i> receptors but had no effect on the interaction with the <i>α3β2</i> subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1’s loop-II interact with <i>α1β1εδ</i>, but only one side interacts with <i>α3β2</i>. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues

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

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

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

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

Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC–FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist–agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist–agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup

Expression of <i>alr</i> during zebrafish embryonic development.

<p>Whole mount in situ hybridization (WISH) shows expression of <i>alr</i> mRNA at different embryonic stages. During early stages, expression of <i>alr</i> is ubiquitous (A, B). Expression in the brain and pharyngeal arches are also observed (C, D, J). From 28 hpf onwards, the expression of <i>alr</i> is detected in liver (white arrow head) throughout hepatogenesis (C–J). C, E, G, I: lateral view, anterior to the left; D, F, H, J: dorsal view, anterior to the left.</p

Knockdown of <i>alr</i> reduces hepatocyte proliferation.

<p>A & B. Hepatocyte proliferation demonstrated by immunofluorescent staining of proliferation markers in 4 dpf embryos: proliferating cell nuclear antigen (PCNA) (A) and phosphor-histone 3 (p-H3) (B). The sections were counterstained with DAPI to label nucleus. PCNA and p-H3 staining is co-localized with DAPI, indicative of nucleus staining. Both PCNA and p-H3 staining showed a significantly reduced hepatocyte proliferation in morphants without affecting proliferation in other tissues such as intestine. I: intestine; L: liver. Dash line circles the boundary of liver. C. Quantification of hepatocyte proliferation. Percentage of PCNA positive hepatocytes in liver is reduced from 13.8% in CO to 6.6% in MO. Percentage of p-H3 positive hepatocytes in liver is reduced from 1.1% in CO to 0.45% in MO. Values are means ± standard deviation (SD). Hepatocytes were counted based on cell morphology.</p