Structural Modeling Predicts Altered Disulfide Connectivity in HER2 W452C

HER2, an ERBB2 -encoded receptor tyrosine kinase, regulates cell growth and survival. HER2 overexpression or ERBB2 amplification can promote tumor growth and is used to identify cancers that may benefit from HER2-targeted therapies. However, the functional consequences of sequence variants are difficult to predict when they occur outside known functional sites. Cysteine-introducing variants pose a challenge because a new cysteine can either form an abnormal bond directly or disrupt an existing disulfide and change which interactions are possible. HER2 W452C variant provides a case study of this problem because it introduces a cysteine outside the canonical dimerization interface but near a native extracellular disulfide. We used structural modeling, molecular dynamics, protein-protein docking, and cell-based analysis to test how W452C could alter receptor organization. Direct C452-C452 pairing between HER2 receptors was consistently difficult to accommodate without severe receptor overlap. We therefore examined a conditional state in which the native disulfide was reorganized, leaving another cysteine available for intermolecular interaction. This state increased ectodomain flexibility and accommodated alternative cysteine-mediated contacts. Explicit-bond refinement showed that selected C452-C504 arrangements remained structurally compatible after covalent disulfide formation. In cells, W452C increased a DTT-sensitive high-molecular-weight HER2-containing species, supporting altered disulfide-dependent association. Together, these findings support a mechanism in which an introduced cysteine alters receptor organization by reorganizing the local disulfide network rather than simply forming a new bond itself. More broadly, they illustrate how structural analysis can reveal molecular consequences of genetic variants that are not apparent from sequence position alone and thereby inform precision-medicine variant interpretation.