Super compensatory substitutions restore protein function and flatten fitness landscapes

Compensatory mutations can mitigate the fitness costs of deleterious mutations, yet their distribution, structural contexts, and evolutionary roles remain poorly characterized. Here, we systematically analyzed a public deep mutational scanning (DMS) dataset comprising ∼400,000 amino acid variants of imidazole glycerol-phosphate dehydratase (IGPD), with substitutions spanning the entire length of the protein. We found that roughly one-third of nonfunctional missense variants can regain function through additional substitutions, predominantly at solvent exposed residues with weak local contacts. Notably, we identified a distinct class of super compensatory substitutions that enhance fitness across diverse genetic backgrounds and buffer mutational effects, effectively flattening the local fitness landscape. Comparative analyses of additional public DMS datasets across diverse proteins and assays revealed that super compensators recur across different natural sequence–function maps, indicating a broadly represented mode of compensatory effects that sustains mutational persistence and contributes to protein evolutionary robustness.