More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions, Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users.Author Summary: Sequence homology is a fundamental principle of biology. It implies common phylogenetic ancestry of genes and, subsequently, similarity of their protein products with regard to amino acid sequence, three-dimensional structure and molecular and cellular function. Originally an esoteric concept, homology with the proxy of sequence similarity is used to justify the transfer of functional annotation from well-studied protein examples to new sequences. Yet, functional annotation via sequence similarity seems to have hit a plateau in recent years since relentless annotation transfer led to error propagation across sequence databases; thus, leading experimental follow-up work astray. It must be emphasized that the trinity of sequence, 3D structural and functional similarity has only been proven for globular segments of proteins. For non-globular regions, similarity of sequence is not necessarily a result of divergent evolution from a common ancestor but the consequence of amino acid sequence bias. In our investigation, we found that protein domain databases contain many domain models with transmembrane regions and signal peptides, non-globular segments of proteins having hydrophobic bias. Many proteins have inherited completely wrong function assignments from these domain models. We fear that future function predictions will turn out futile if this issue is not immediately addressed.