Using evolutionary context to classify difficult protein folds

Recent advances in protein structure prediction, such as AlphaFold2, have enabled identification of vast numbers of putative novel protein domains across the sequence space, many of which adopt structures dissimilar to known folds. Based on structural segmentation and classification, the Encyclopedia of Domains (TED) project recently cataloged more than 7400 low-symmetry, structure-based domains as candidate novel-fold (CNF) domains. To place these domains in their broader evolutionary and structural context, we applied DPAM (Domain Parser for AlphaFold Models), a complementary method that combines AlphaFold-derived confidence metrics with sensitive sequence and structure similarity searches, to parse domains for the AlphaFold models of proteins containing TED CNF domains. We identified 8044 DPAM domains with significant overlap with TED CNF domains, among which 2490 were confidently assigned to entries in the ECOD (Evolutionary Classification of protein Domains) structural classification hierarchy. Our results suggest that a substantial subset of TED candidate novel-fold domains are distant homologs of existing ECOD domains. Comparison of domain boundaries between TED and DPAM showed varied patterns: more than one-third of cases featured TED CNF domains largely embedded within DPAM domains-often representing insertions or extensions into enzymatic or repeat folds. A smaller fraction (17%) exhibited consistent domain boundaries between TED and DPAM. These consistently defined domains are often characterized by significant structural diversity, including long insertions and duplications. An even smaller subset showed the reverse relationship, with DPAM domains largely embedded within TED CNF domains. In these cases, DPAM effectively separated multiple structural units that TED grouped as single domains. Together, these findings highlight the complementarity of structural and evolutionary approaches for domain annotation and demonstrate the power of integrative methods, such as DPAM, in refining the classification of challenging protein folds and uncovering distant evolutionary relationships.