Conversion of MIG6 peptide from the nonbinder to binder of lung cancer-related EGFR by phosphorylation and cyclization

Figures & data

Figure 1. The complex crystal structure of EGFR kinase domain with MIG6^s1 and MIG6^s2 (PDB: 4ZJV).

Figure 2. (A) The phosphorylated MIG6^s2 peptide was stripped from its cocrystallizated complex structure with EGFR kinase domain (PDB: 4ZJV), resulting in unbound phosphorylated peptide with the conformation as it in cocrystallizated complex. (B) The stripped peptide was dephosphorylated at residues pTyr394 and pTyr395 without conformation change, resulting in a dephosphorylated version of the peptide. (C) The Leu397 residue of the stripped peptide was virtually mutated to Cys397, which was then covalently connected to wild-type Cys378 residue by computationally modeling a disulfide bond between them, resulting in a phosphorylated cyclic peptide. Subsequently, the three artificially modeled peptides were subjected to MD simulations to reach at equilibrium state.

Table 1. The calculated energetics parameters and experimental affinities for the different forms of MIG6^s2 peptides to interact with EGFR kinase domain.

MIG6^s2		Calculated energetics (kcal/mol)				Experimental affinity Kd (μM)
phosphorylation^a	cyclization^b	ΔEint	ΔDdslv	−TΔS	ΔG
no	no	−176.4	115.8	67.8	7.2	n.d.^c
yes	no	−240.7	160.9	76.4	−3.4	95 ± 7
yes	yes	−226.2	175.7	34.2	−16.3	11.2 ± 1.4

^a yes, both the Tyr394 and Tyr395 residues of MIG6^s2 peptide are phosphorylated; no: both the Tyr394 and Tyr395 residues of MIG6^s2 peptide are dephosphorylated.

^b yes, the Leu397 residue of MIG6^s2 peptide is mutated to Cys397 and then covalently connected to wild-type Cys378 residue via a disulfide bond; no: no mutation and connection are addressed on the MIG6^s2 peptide.

^c n.d., not determined.

Figure 3. Cyclization of phosphorylated MIG6^s2 peptide. (A) The phosphorylated peptide was stripped from its cocrystallizated complex with EGFR kinase domain (PDB: 4ZJV). (B) The Leu397 residue of the peptide was virtually mutated to Cys397 (L397C). (C) The peptide was cyclized by introducing a disulfide bond between the mutated Cys397 and wild-type Cys378.

Figure 4. Titration of recombinant EGFR kinase domain to different chemical forms of fluorescein-labeled MIG6^s2 peptides.