Abstract PS3-06-28: Identification of APOBEC3 Protein Sectors in Cancer Through Evolutionary Conservation and Co-variation Analysis

Abstract The functionality of proteins is intrinsically linked to their structural composition. The primary structure refers to the linear sequence of amino acids. Secondary structures, such as α-helices and β-sheets, arise from hydrogen bonding within the polypeptide backbone. These elements fold into the tertiary structure, defining the protein's three-dimensional shape. Beyond these traditional classifications, the concept of protein sectors offers a dynamic view of protein functionality. Protein sectors consist of co-evolving amino acid groups, highlighting functional interdependence. Human DNA codes for 11 Cytidine deaminases including 7 APOBEC3 enzymes known for their roles in DNA mutagenesis. These enzymes play a critical role as antiviral proteins but their aberrant activity has been shown to lead to mutations in cellular DNA leading to cancer. APOBEC3 enzymes are multifunctional proteins, however, currently, it is unclear how amino acid residues of these enzymes have co-evolved and whether they form distinct sectors, each responsible for a specific function. The mechanisms underlying functional specificity of members of this enzyme family are also mostly unknown. For example, APOBEC3A and APOBEC3B are localized in the nucleus, and are involved in inhibiting the replication of endogenous elements and exogenous DNA viruses. In contrast, A3D/F/G/H are found in the cytoplasm, primarily targeting RNA viruses, especially retroviruses. It's plausible that the unique localization of APOBEC3A and APOBEC3B is influenced by a specific sector that enables nuclear localization, possibly through interaction with unique cellular proteins. Here, we introduce a novel application of statistical coupling analysis (SCA) to identify APOBEC3 protein sectors and elucidate their functional roles. SCA identifies pairs of amino acid positions that co-vary across a set of homologous proteins, indicating functional or structural importance. SCA applies spectral analysis to a matrix combining correlation information with sequence conservation, revealing patterns of correlated mutations. The significant co-evolved sections are distinguished by eigenvector decomposition, helping to understand the evolutionary dependencies and functional interdependencies within proteins, providing insights into protein structure and function. Using this methos, we recently analyzed 5000 unique sequences (APOBEC1, APOBEC2, APOBEC3A/B/C/D/F/G/H, APOBEC4, AID) from a diverse range of species. Our analysis identified five distinct protein sectors. Mapping co-evolving sectors onto the APOBEC3A reference structure (PDB ID: 4XXO) revealed distinct functional compartmentalization among key catalytic and DNA-binding residues. Specifically, sector 2 exclusively encompassed the zinc-coordinating triad—His70, Cys101, and Cys103—highlighting its likely role in enzymatic catalysis. In contrast, sector 4 clustered residues Asn57, Trp98, and Tyr130, which are known to mediate substrate DNA recognition and binding. Ongoing analyses aim to characterize additional sectors that may modulate substrate specificity, mutagenic intensity, and/or regulatory interactions.In conclusion, given the multifunctional nature of APOBEC enzymes, it is crucial to discern, using a systematic evolutionary approach, how functions like DNA binding, RNA binding, deaminase activity, cellular localization, and other unknown roles have evolved distinctly in APOBEC proteins, and which amino acids play a part in each functional domain. Citation Format: N. Haghjoo. Identification of APOBEC3 Protein Sectors in Cancer Through Evolutionary Conservation and Co-variation Analysis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-06-28.