Y669C

Position 669 sits in wolframin's C-terminal lumenal domain (residues 653–869). The AlphaFold model places Y669 within 5 Å of GLN668 (2.5 Å), GLY670 (2.5 Å), TRP666 (3.8 Å), THR665 (4.0 Å), ALA671 (4.4 Å), and GLN667 (4.5 Å). The wild-type tyrosine ring sits in an aromatic-adjacent environment — TRP666 at 3.8 Å is consistent with edge-face π–π stacking between the Y669 phenol and the W666 indole. The hydroxyl group of the wild-type also makes plausible hydrogen-bonding contacts to GLN668 and THR665.

Replacing tyrosine with cysteine at this position has three layered effects. First, the aromatic ring is gone, eliminating the π-stacking with TRP666 and the hydrophobic surface contributed to the local fold. Second, the hydroxyl group is gone, removing two hydrogen-bond contacts. Third, a reactive free thiol is introduced into the oxidizing ER lumen — and the lumenal domain contains other cysteine residues (notably CYS673 at 3.8 Å from C690, a separate position) that could engage in aberrant disulfide formation. The new C669 is not directly in contact with another cysteine in the AlphaFold model, but disulfide chemistry in an oxidizing compartment is promiscuous.

DynaMut2 returns a modest |ΔΔG| of 0.41 kcal/mol. The fold survives the substitution. The damage is mechanistic — lost aromatic packing, lost H-bonding, plus the off-pathway disulfide risk that DynaMut2's structural prediction cannot fully capture.