Figure 1.
Identification of the mutation sites on the decoy receptor, TV3, for constructing single variants.
(A) Crystal structure of the decoy receptor in complex with MD2 (PDB ID 2Z65). The decoy receptor and MD2 are shown as green and yellow, respectively. (B) Structure of the interaction interface. 14 identified residues in TV3 are indicated in green, and potential interaction residues in MD2 are represented in yellow.
Table 1.
Binding affinities of single mutants for MD2.
Figure 2.
Crystal structure of the F63W mutant in complex with MD2.
(A) Superimposed backbone structure of F63W mutant/MD2 complex into the wild-type decoy receptor/MD2 complex structure. The F63W showed a slight movement toward the N-terminal direction by 0.4 Å compared to the wild-type decoy receptor in complex with MD2. (B) The complex structure of F63W mutant/MD2. The mutated Tyr-63 was closely located to Arg-68 on MD2, creating cation-π interaction. The F63W mutant in complex structure is indicated as red and MD2 of F63W/MD2 complex is colored in yellow. The electron density map of mutant complex is shown as blue. (C) Comparison of the wild-type decoy receptor/MD2 and F63W/MD2 complex structures. The key residues and backbone structure of wild-type decoy receptor/MD2 complex are shown in grey. In F63W/MD2 complex, the F63W mutant is shown in red, and MD2 in yellow, respectively.
Figure 3.
Interface structures of single variants.
(A) Superimposed backbone structures of the wild-type decoy receptor and three single mutants (M41E, F63W, and V134L). (B) Interface structure of the M41E mutant obtained by superimposing the crystal structure of apo M41E mutant into the wild-type decoy receptor/MD2 complex structure. Glu-41 was positioned to be able to form a salt-bridge with Lys-109 on MD2. The crystal structure of the M41E is shown in blue, and the wild-type decoy receptor and MD2 are colored in green and yellow, respectively. (C) Interface structure of the V134L mutant obtained by superimposing the crystal structure of apo V134L mutant into the wild-type decoy receptor/MD2 complex structure. The larger Leu-134 is possible to make closer contact with Leu-108 on MD2. The crystal structure of the V134L is colored in orange (D) Interface structure of the F63W mutant obtained by superimposing the crystal structure of apo F63W mutant into the wild-type decoy receptor/MD2 complex structure. The mutated Trp-63 was expected to create the amino-aromatic (cation-π) interaction of Tyr-42 and Arg-68. The crystal structure of the F63W is shown in red.
Table 2.
Binding affinities of double mutants for MD2.
Table 3.
Changes in the interaction energies and hydrogen bond numbers.
Figure 4.
Snapshots of molecular dynamics simulation trajectories.
Each snapshot was chosen to represent the changes in interaction energies. (A) Modeled structure of the double mutant, M41E/F63W. Increased hydrophobic environment resulting from the F63W mutation induced strong charge interactions between Glu-41 of the decoy receptor and Arg-68, Arg-69, and Lys-109 on MD2. (B) Modeled structure of the double mutant, V134L/H159Q. The V134L mutation led to increased hydrophobic interaction, strengthening the charge interaction between Gln-159 on decoy receptor and Glu-111 on MD2.