Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States

The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein

Optimization of structures using the ONIOM method.

<p>Residues of IsdH-N3, IsdA-N, and IsdC-N are shown in cyan, yellow, and orange, respectively. Heme is shown in purple. (<b>A</b>) IsdH–Tyr642 and IsdA–Tyr170 are deprotonated in the IsdH-N3•heme•IsdA-N complex and (<b>B</b>) IsdA–Tyr166 and IsdC–Tyr132 are deprotonated in the IsdA-N•heme•IsdC-N complex.</p

MD simulations of Isd•heme•Isd ternary complexes.

<p>(A and D) Overall structures of the docked complexes IsdH-N3, IsdA-N, IsdC-N, and heme are represented in cyan, yellow, orange, and purple, respectively. The IsdH-N3•heme•IsdA-N and IsdA-N•heme•IsdC-N snapshots were obtained in MD simulations at 1,000 and 900 ns, respectively. (B and E) Plots of distances in MD trajectories; black, green, red, and blue traces represent distances between side-chain Oγ atoms of conserved serine residues and proximal carboxyl groups of heme, between Oη atoms of the primary tyrosine residues and iron, and between the secondary tyrosine and iron, respectively. (C and F) Plots of RMSD values; crystal structures were used as reference structures. Panel F: RMSD values that were calculated excluding the β7-β8 hairpin in IsdC-N (residues Asp-118 to Tyr-136) are presented in the blue trace.</p

Superposition of IsdH-N3 (cyan), IsdB-N2 (green), IsdA-N (yellow), and IsdC (orange) crystal structures.

<p>(A) Overview of Isd–NEAT domains; the heme molecule and primary/secondary tyrosine residues on the β8 strand are represented as ball-and-stick models; the iron atom of heme is shown as a green sphere. (B) Close-up view of the heme-binding pocket; heme propionate groups form H-bonds with conserved serine residues between the loop 1 region and the 3₁₀-helix (IsdH–Ser563, IsdB–Ser361, IsdA–Ser82, or IsdC–Ser47).</p

Optimized structures with an additional proton.

<p>Residues of IsdH-N3, IsdA-N, and IsdC-N are shown in cyan, yellow, and orange, respectively. Heme is shown in purple. (A) Protonated IsdH–Tyr642 and–Tyr646; (B) protonated IsdA–Tyr166 and–Tyr170 in the IsdH-N3•heme•IsdA-N system; (C) protonated IsdA–Tyr166 and–Tyr170 in the IsdA-N•heme•IsdC-N system; (D) protonated IsdA–His83 and IsdC–Tyr136; an additional proton was initially placed near the Nδ atom of IsdA–His83.</p

Click Conjugation of a Binuclear Terbium(III) Complex for Real-Time Detection of Tyrosine Phosphorylation

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb­(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb­(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb­(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis–Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery

MD simulations of Isd•heme•Isd ternary complexes.

<p>(A and D) Overall structures of the docked complexes IsdH-N3, IsdA-N, IsdC-N, and heme are represented in cyan, yellow, orange, and purple, respectively. The IsdH-N3•heme•IsdA-N and IsdA-N•heme•IsdC-N snapshots were obtained in MD simulations at 1,000 and 900 ns, respectively. (B and E) Plots of distances in MD trajectories; black, green, red, and blue traces represent distances between side-chain Oγ atoms of conserved serine residues and proximal carboxyl groups of heme, between Oη atoms of the primary tyrosine residues and iron, and between the secondary tyrosine and iron, respectively. (C and F) Plots of RMSD values; crystal structures were used as reference structures. Panel F: RMSD values that were calculated excluding the β7-β8 hairpin in IsdC-N (residues Asp-118 to Tyr-136) are presented in the blue trace.</p

Proposed heme transfer mechanism from IsdH-N3 to IsdC via IsdA.

<p>The sides of heme are shown in black and white. Heme is inverted upon formation of the complex between acceptor and donor NEAT domains.</p

Two-dimensional potential energy surface of the system of two phenols and an Fe(III)-porphine.

<p>Black contour lines are at intervals of 3 kcal/mol.</p

Supplementary data from Haemolytic actinoporins interact with carbohydrates using their lipid-binding module

Table S1. Structural homology of FraC; Figure S1. Kinetics of hemolysis by FraC in the presence of saccharides; Figure S2. Depiction of the lipid/carbohydrate binding region of FraC; Figure S3. Comparison of FraC with a fungal lectin