Dynamic consequences of threonine mutations in the CaLM motif of RET/GFRα1/GDNF ternary complex

The extracellular domain (ECD) of the RET receptor tyrosine kinase depends on its cysteine-rich domain (CRD) for calcium coordination, structural stability, and assembly with GFRα1 and GDNF. Mutations close to the CRD CaLM motif have been associated with disease, but their molecular effects remain understudied. In this study, we analyzed two clinically reported variants, T564N and T564P, using all-atom molecular dynamics simulations of both the isolated CRD and the RET/GFRα1/GDNF ternary complex. Our analysis showed that both the mutations introduced localized structural changes in the CRD monomer. T564N caused increased residue fluctuations at the mutation site and solvent exposure, whereas T564P enhanced flexibility across all calcium-coordinating residues and slightly decreased stabilizing contacts. These effects became more noticeable in the ternary complex. Within the complex, interactions with the neighbouring domains caused the CRD to adopt conformations that compensated for the structural changes observed in the CRD monomer. In this context, each mutation affected calcium-binding energetics differently, resulting in more favourable binding in the mutants than in the wild-type. Although calcium binding was energetically favourable, the overall interaction energy within the complex was still affected. The complex highlighted mutation-specific differences in RET's interactions with GFRα1 and GDNF. The comparison between monomeric and complex simulations indicates that the functional impact of T564 mutations cannot be inferred from the isolated CRD. Together, these results show that the structural and energetic consequences of CRD CaLM mutations depend strongly on the full signaling assembly. This underscores the need to assess RET variants within their native multiprotein environment to understand how disease-associated mutations may alter receptor function.