The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant.

RET（转染过程中重新排列）是一种跨膜酪氨酸激酶，其异常激酶活性与人类癌症有关。由位于活化环（A-环）上的溶剂前亲水性 S904 替换为大的疏水性苯丙氨酸残基引起的点突变可诱导对 I 型激酶抑制剂 vandetanib 的抗性。这种耐药性的一个可能机制是释放顺式- RET 的自抑制构象用于自磷酸化，从而激活 RET 激酶。由于 S904F 突变与增强的自磷酸化之间的关联尚不清楚，我们进行了分子建模分析以比较未磷酸化的 apo 野生型和 S904F 突变体结构。A 环的结构紧凑性促进了 ATP 结合。当 A 环延伸时，αC 螺旋向富含甘氨酸的环移动，导致 F735 突出。挤压的 F735 与 E734 和 R912 连接并约束 ATP 口袋入口。相反，一个收缩的 A 环将 αC 螺旋从富含甘氨酸的环中拉开，掩埋 F734 并使 ATP 口袋可以进入。突变的 F904 稳定了收缩的 A 环并释放了 RET 的自抑制构象，从而促进自磷酸化。我们还模拟了两个 ATP 结合系统。ATP 的结合自由能，通过分子力学和广义的波恩和表面积溶剂化方法估计，表明 S904F 突变体比野生型与 ATP 的结合更紧密。这些发现支持 S904F 突变体中自磷酸化促进的前提。