Exploring the Terra incognita of AI-based domain classifications

The classification of novel protein folds remains a central challenge in structural bioinformatics, particularly as deep learning models like AlphaFold2 dramatically expand the universe of predicted protein structures. In this study, we investigated 664 candidate novel fold (CNF) domains from the TED database that both TED and DPAM methods had classified with low confidence. These CNFs span a structurally diverse and largely non-redundant set of domains, most of which lack clear sequence or structural similarity to known folds. Many CNFs appear as insertions into known transmembrane or enzymatic domains, while others occur in modular architectures, co-occurring with interaction or catalytic folds such as β-barrels, zinc fingers, or Rossmann-like domains. Although some CNFs resemble known folds that have undergone topological rearrangements or circular permutations, others result from errors in domain boundary prediction, often due to truncated sequences or tightly packed domain duplications. Our analyses led to the creation of 190 new Pfam families, many classified as domains of unknown function (DUFs), and revealed intriguing cases of zinc-binding and disulfide-rich architectures that contribute to fold space expansion. A small subset of CNFs helped define new superfamilies by linking previously unclassified but structurally related domains. Taken together, this work underscores the importance of integrating structural, evolutionary, and contextual information to resolve challenging fold assignments and provides a roadmap for extending protein classification frameworks into previously uncharted structural territory.