Computational engineering of water-soluble human potassium ion channels through QTY transformation

Figures

• 

  Fig. 1. Protein alignments of 6 Cryo-EM structure potassium ion channels with their water-soluble QTY variants. The symbols | and * indicate the identical and different amino acids, respectively. Please note the Q, T and Y amino acid replacement (red). The alpha-helices (blue) are shown above the protein sequences. The loop color codes are internal (yellow) and external (red). Features of natural and QTY variants are pI, molecular weight (MW), total variation (%) and transmembrane variation (%). The alignments are (a) KCNN4 vs KCNN4QTY, (b) KCNK2 vs KCNK2QTY, (c) KCNA3 vs KCNA3QTY, (d) KCNMA1 vs KCNMA1QTY, (e) KCNH2 vs KCNH2QTY, (f) KCNJ11 vs KCNJ11QTY. Although there are significant QTY changes for the TM alpha-helix changes, > 48.4% for KCNN4, > 60% for KCNK2, their changes of molecular weight and pI are insignificant, two have no pI changes at all (KCNA3 and KCNMA1) (Table 2).

• 

  Fig. 2. Protein alignments of 12 AlphaFold 2 predicted native potassium ion channels with their water-soluble QTY variants. All characteristics and color codes are referred in Fig. 1. The alignments are (a) KCNA1 vs KCNA1QTY, (b) KCNA5 vs KCNA5QTY, (c) KCNH5 vs KCNH5QTY, (d) KCNJ3 vs KCNJ3QTY, (e) KCNJ8 vs KCNJ8QTY, (f) KCNK5 vs KCNK5QTY, (g) KCNK9vs KCNK9QTY, (h) KCNC4 vs KCNC4QTY, (i) KCND1 vs KCND1QTY, (j) KCNJ10 vs KCNJ10QTY, (k) KCNJ12 vs KCNJ12QTY, (l) KCNN3 vs KCNN3QTY. Although there are significant QTY changes for the TM alpha-helix changes, > 50% for KCNH5, ~ 57% for KCNA1, their changes of pI and molecular weight (MW) are insignificant and some have no pI changes at all (KCNA1, KCNA5, KCNK5, KCNC4, and KCNJ12) (see Table 2).

• 

  Fig. 3. Superposed 6 potassium ion channels Cryo-EM structures with QTY variants predicted by AlphaFold2. The Cryo-EM structures of the native transporters are obtained from the Protein Data Bank (PDB). The Cryo-EM structures (magenta) are superposed with QTY variants (cyan) predicted by AlphaFold2. KCNN4Cryo-EM vs KCNN4QTY (1.790 Å), KCNK2Cryo-EM vs KCNK2QTY (3.358 Å), KCNA3Cryo-EM vs KCNA3QTY (2.236 Å), KCNMA1Cryo-EM vs KCNMA1QTY (1.474 Å), KCNH2Cryo-EM vs KCNH2QTY (2.077 Å), KCNJ11Cryo-EM vs KCNJ11QTY (0.944 Å). These superposed structures display that Cryo-EM structures and their AlphaFold2 predicted native transporters have very similar molecular structures. For clarity of direct comparisons, the N-terminus and C-terminus are removed.

• 

  Fig. 4. Superposed 6 potassium ion channels Cryo-EM structures with native structures predicted by AlphaFold2. The Cryo-EM structures of the native transporters are obtained from the Protein Data Bank (PDB). The Cryo-EM structures (magenta) are superposed with native structures (green) predicted by AlphaFold2. The RMSD (Å) for each structure are: KCNN4Cryo-EM vs KCNN4AF2 (1.779 Å), KCNK2Cryo-EM vs KCNK2AF2 (2.146 Å), KCNA3Cryo-EM vs KCNA3AF2 (1.449 Å), KCNMA1Cryo-EM vs KCNMA1AF2 (0.847 Å), KCNH2Cryo-EM vs KCNH2AF2 (1.953 Å), KCNJ11Cryo-EM vs KCNJ11AF2 (0.617 Å). These superposed structures display that Cryo-EM structures and their AlphaFold2 predicted transporters have very similar molecular structures. For clarity of direct comparisons, the N-terminus and C-terminus are removed.

• 

  Fig. 5. Superposed native potassium ion channels and their QTY variants that were predicted by AlphaFold2. The native structures (green) and their water-soluble variants (cyan). For clarity, N-terminus, C-terminus and large loops are deleted. KCNA1 vs KCNA1QTY (1.565 Å), KCNA3 vs KCNA3QTY (1.092 Å), KCNA5 vs KCNA5QTY (1.551 Å), KCNH5 vs KCNH5QTY (1.543 Å), KCNJ3 vs KCNJ3QTY (1.254 Å), KCNJ8 vs KCNJ8QTY (0.540 Å), KCNK2 vs KCNK2QTY (2.093 Å), KCNK5 vs KCNK5QTY (0.967 Å), KCNK9 vs KCNK9QTY, (0.514 Å), KCNC4 vs KCNC4QTY (1.105 Å), KCND1 vs KCND1QTY (1.025 Å), KCNH2 vs KCNH2QTY (1.547 Å), KCNJ10 vs KCNJ10QTY (1.728 Å), KCNJ11 vs KCNJ11QTY (0.446 Å), KCNJ12 vs KCNJ12QTY (0.397 Å), KCNMA1 vs KCNMA1QTY (0.981 Å), KCNN3 vs KCNN3QTY (1.917 Å), KCNN4 vs KCNN4QTY (1.003 Å). Please find all RMSD values (Å) in Table 2.

• 

  Fig. 6. Hydrophobic surface of 18 potassium ion channels and the designed QTY variants. The native solute carrier transporters have many hydrophobic residues L, I, V and F in the transmembrane helices (yellow color). After Q, T, and Y substitutions of L, I, V, F, the protein surfaces become more hydrophilic (cyan color). Overall, the hydrophobic surface (yellow) of the native transporters become more hydrophilic (cyan). The hydrophobic surface is largely reduced on the transmembrane helices for the QTY variants. These QTY variants converted to water-soluble form. For clarity, the N-terminus and C-terminus are deleted.

• 

  Fig. 7. Bioinformatic comparison of protein sequence characteristics between native, Cryo-EM, and QTY structures. Grand average of hydropathy (GRAVY) and solvent-accessible surface area (SASA) are important indicators for protein solubility. A GRAVY value < 0 implies a higher degree of hydrophilicity in a protein, while a GRAVY value > 0 indicates a hydrophobic nature. Higher flexibility values reflect greater flexibility. Solubility values below 0.5 correspond to insoluble proteins, while values above 0.5 show soluble proteins. (a) Kruskal–Wallis H-test was performed between native and QTY, native, Cryo-EM, and QTY proteins to compare the differences in stability, GRAVY, flexibility, SASA, and solubility. The red line reflects p-value < 0.05 (significance level). The Kruskal–Wallis H-test is a non-parametric statistical test used to compare the medians of three or more independent groups (on the figure, the groups are stability, GRAVY, flexibility, SASA, and solubility), assessing whether there are significant differences among them. Significant differences between native and QTY proteins can be observed in GRAVY, flexibility, and solubility, while stability and SASA remain similar. (b)…

• 

  Fig. 8. Bioinformatic comparison of protein inter-atomic interactions between native, Cryo-EM, and QTY variant structures. Hydrogen bonds (HBO), polar contacts (PLR), ionic interactions (ION), aromatic contacts (ARO), hydrophobic contacts (HDP), van der Waals interactions (VDW), and van der Waals clashes (VCL). HBO are electrostatic attractive forces between hydrogen covalently bound to a more electronegative donor and electronegative acceptor. PLR refer to interactions between polar groups of amino acids. ION involve the attraction between oppositely charged ions. ARO exist in interactions between aromatic rings of phenylalanine, tryptophan and tyrosine. HDP are interactions when hydrophobic groups of amino acids cluster together. VDW are caused by weak attractive forces between nonpolar atoms. VCL represents steric repulsion due to the overlap of van der Waals radii. (a) Kruskal–Wallis H-test was performed between i) native and QTY, ii) native and Cryo-EM, and iii) Cryo-EM and QTY proteins to compare the differences in HBO, PLR, ION, ARO, HDP, VDW, and VCL. The red line reflects p-value < 0.05 (significance level). The Kruskal–Wallis H-test is a non-parametric statistical test used to…

• 

  Fig. 9. Convergence and stability of MD simulations. We performed molecular dynamics (MD) simulations of 2 proteins shown best in the interaction analysis based on rigid structures (KCNJ11 and KCNN4). In sum, dynamic analysis was made on the converged part of 8 MD systems: KCNN4Cryo-EM, KCNN4Native, KCNN4QTY in membrane, KCNN4QTY in water, KCNJ11Cryo-EM, KCNJ11Native, KCNJ11QTY in membrane, and KCNJ11QTY in water. (a) The converged part is marked by the red line (starting from 300 ns and frame #3,000 for proteins in a membrane, and 200 ns and frame #2,000 for proteins in water). We put the corresponding mean and standard deviation of RMSD values at this part of trajectories above the red line. RMSD values were calculated relative to the first simulation frame of each system. (b) Protein conformations at the beginning (frame #0, 0 ns) and at the end (frame #4,000, 400 ns for proteins in a membrane and frame #3,000, 300 ns for proteins in water) of MD simulations. Each image depicts one variant (Cryo-EM, native, or QTY) of the protein (KCNN4 or KCNJ11) in a membrane (gray lipids with orange phosphates at the membrane edges), surrounded by water and ions (Cl- is green and K+ is violet). All…

• 

  Fig. 10. Bioinformatic comparison of protein inter-atomic interactions between native, Cryo-EM, and QTY variant structures through MD simulations. We performed molecular dynamics (MD) simulations of 2 proteins shown best in the interaction analysis based on rigid structures (KCNJ11 and KCNN4). In sum, dynamic analysis was made on the converged part of 8 MD systems: KCNN4Cryo-EM, KCNN4Native, KCNN4QTY in membrane, KCNN4QTY in water, KCNJ11Cryo-EM, KCNJ11Native, KCNJ11QTY in membrane, and KCNJ11QTY in water. Other abbreviations mean hydrogen bonds (HBO), polar contacts (PLR), ionic interactions (ION), aromatic contacts (ARO), hydrophobic contacts (HDP), van der Waals interactions (VDW), and van der Waals clashes (VCL). HBO are electrostatic attractive forces between hydrogen covalently bound to a more electronegative donor and electronegative acceptor. PLR refer to interactions between polar groups of amino acids. ION involve the attraction between oppositely charged ions. ARO exist in interactions between aromatic rings of phenylalanine, tryptophan and tyrosine. HDP are interactions when hydrophobic groups of amino acids cluster together. VDW are caused by weak attractive forces between…

• 

  Fig. 11. Structural comparison of native, Cryo-EM, and QTY variant conformations from MD simulations. We performed molecular dynamics (MD) simulations of 2 proteins which showed best in the interaction analysis based on rigid structures (KCNJ11 and KCNN4). In sum, dynamic analysis was made on the converged part of 8 MD systems: KCNN4Cryo-EM, KCNN4Native, KCNN4QTY in membrane, KCNN4QTY in water, KCNJ11Cryo-EM, KCNJ11Native, KCNJ11QTY in membrane, and KCNJ11QTY in water. (a) Structural alignment between the conformations from the start (gray; 0 ns, frame #0) and end (main variant color; 400 ns, frame #4,000 for proteins in membrane and 300 ns, frame #3,000 for proteins in water) conformations of each trajectory. (b) Structural alignment between Cryo-EM conformations and other variant conformations. Each alignment includes 20 converged conformations (10 per system). (c) General distribution of RMSD values calculated based on pairwise comparison of all converged conformations for each pair of systems.

• 

  Fig. 12. Structural analysis of oligomer interfaces formed by QTY variants. We superimposed the last MD conformations of KCNN4Cryo-EM, KCNN4Native, KCNN4QTY in membrane, KCNN4QTY in water, KCNJ11Cryo-EM, KCNJ11Native, KCNJ11QTY in membrane, and KCNJ11QTY in water monomers on the original Cryo-EM oligomers from PDB. Similar to the previous analysis, the Cryo-EM structures are colored in magenta (both monomers and oligomers), QTY variants are cyan, and native structures are green. (a) The placement of native, QTY, and Cryo-EM monomers in the Cryo-EM oligomer. QTY models fit well into the original Cryo-EM oligomer, though form a small fraction of the oligomer assemblies. (b) Structural interface and positions of the QTY mutations on the interface. The interface is defined as monomer residues that are placed within 5 Å distance from other domains. C-alpha atoms of the interface residues are shown as spheres and the QTY-mutated residues are colored in black. There are only a few QTY mutations on the monomer interfaces, implying no major impact on the functional oligomer.