Systematic Disagreement between ESMFold and MSA-Based Structure Predictors on Chimeric Fusion Proteins

Single-sequence structure predictors such as ESMFold are widely used as computational filters for designed proteins, including multi-domain fusions absent from natural sequence databases. Whether this filter agrees with MSA-based predictors on such constructs has not been established. We assemble 1,000 chimeric domain pairs that do not occur in UniProt, held out by protein family, and score each fusion by the worse domain's TM-score against its native AlphaFold Database conformation. ESMFold exhibits a strong, internally reproducible preference for one domain order (Pearson r=0.90 under composition-matched linker swaps) that Boltz-2 does not share (r=0.07). On an unbiased subset of 300 constructs, ESMFold assigns TM<0.7 to 17.7% of fusions and Boltz-2 to 8.7%, with only ten shared failures. AlphaFold 2, on the same alignments, fails on 7/10 of those consensus failures and on 1/43 of the failures unique to ESMFold. Isolated-domain controls partition the latter set: 26 constructs contain a domain that ESMFold already fails alone—domains that isolated Boltz-2 and AlphaFold 2 both fold—and 17 fail only after fusion. The fusion-dependent failures coincide with elevated drift in the affected domain's pair block, recur across unrelated partners, and a held-out dummy fusion flags them at high precision. On the six ESMFold-only domains with deposited crystal structures, Boltz-2 and AlphaFold 2 agree with the crystal (mean TM 0.88 and 0.89) while ESMFold does not (0.22). Neither a stricter TM threshold nor an ESMFold confidence gate resolves the disagreement. MSA models recover a population-level advantage of natural structured junctions over a glycine-serine linker, but do not agree on which pairs benefit. Predictor scores are therefore not interchangeable on artificial fusions; the claim concerns the predictors, not the proteins.