Structural and functional roles of conserved residues of human papillomavirus (HPV) E2 protein and biological consequences

Abstract

Background: Human papillomavirus (HPV) is a prevalent viral pathogen that causes a variety of malignancies, including cervical cancer, one of the leading causes of cancer-related deaths among women worldwide. The HPV E2 protein is a central regulator of viral replication and oncogene expression, making it a critical determinant of HPV-associated malignancies. While its core functions are conserved, variations within the E2 protein are thought to contribute to the differential oncogenic potential among HPV types, though the structural basis for this remains incompletely understood. Previous research from our laboratory suggests that mutations within a 12-base pair segment of the long control region that encompasses the E2 binding sites may influence the oncogenic potential of certain HPV strains.

Methods: Computational methods, including multiple sequence alignment, phylogenetic analysis, and protein structural modeling were employed to identify conserved regions and correlate these with potential cancer-associated mutations in the coding region.

Results: Structural modeling using AlphaFold3 and visualization in PyMOL revealed that conserved E2 residues cluster near the DNA-binding surface in the C-terminal domain and at critical interaction sites in the N-terminal transactivation domain, important for E1 DNA helicase binding and potentially other host factor interactions. Notably, species-specific adaptations, including the T309P substitution in the HPV52 subfamily B2, which may induce structural changes in the DNA-binding domain, and variations in the 12-base pair spacer, could modulate oncogene expression.

Conclusions: Collectively, these findings refine our understanding of E2's essential role in viral pathogenesis and highlight promising targets for therapeutic intervention in high-risk HPV strains.

Keywords: DNA-binding domain; E2 protein; Human papillomavirus; Oncogenic potential; Sequence conservation; Structural modeling; T309P mutation; Transactivation domain.

Fig. 1

Genome maps of representative HPV genera. Genome map of three representative genera: A. HPV18 (Alphapapillomavirus), B. HPV49 (Betapapillomavirus), C. HPV4 (Gammapapillomavirus). In A, the HPV18 genome map includes an enhanced view of the E2-LCR DNA interaction, highlighting the positioning of E2 binding sites (E2BS) within the Long Control Region (LCR) and their spatial arrangement relative to the E6 and E7 oncogenes. Created using BioRender & Genome maps retrieved from PAVE

Fig. 2

Phylogenetic tree of HPV E2 protein sequences. Phylogenetic tree of 215 HPV E2 protein sequences, illustrating the evolutionary relationships among Alpha (red), Beta (blue), and Gamma (green) papillomaviruses. The tree highlights distinct clades within each genus, with a scale bar indicating genetic distance. A complete list of all 215 strains included in the analysis is provided in Additional file 1. Created using BioRender

Fig. 3

Schematic of E2 protein domains in representative HPV genera. Schematic of the E2 protein domains in three representative HPV genera: (A) HPV-18 (α), (B) HPV-49 (β), and (C) HPV-4 (γ). The N-terminal transactivation domain (orange) and C-terminal DNA-binding domain (blue) are shown, separated by a flexible linker region whose length in amino acids (AA) is indicated. The number of conserved residues out of the total amino acids within each domain (e.g., 36/201 AA) is indicated parenthetically. Created using BioRender

Fig. 4

AlphaFold prediction model of the HPV-18 E2 protein. The top panel shows the full-length predicted structure with the N-terminal transactivation domain (TAD) colored dark blue, and the C-terminal DNA-binding domain (DBD) colored cyan, connected by a flexible linker region (green). Enhanced views show: (A) the C-terminal DNA-binding domain (DBD) and (B) the N-terminal transactivation domain (TAD). Predicted TM-scores (pTMscore) for the individual domains are 0.85 (A - DBD) and 0.91 (B - TAD). Conserved residues identified from multiple sequence alignments are highlighted in red. Key α-helices are labeled sequentially within each domain

Fig. 5

Residue-Nucleotide Contact Maps for HPV E2-DNA Complexes. The diagrams illustrate individual nucleotide-residue interactions for (A) HPV18 E2 protein (PDB: 1JJ4) and (B) HPV6 E2 protein (PDB: 2AYB) with their respective palindromic E2 binding site (E2BS) targets. Protein residues (small nodes with shape/color indicating secondary structure) from the E2 recognition helix are shown making hydrogen-bonding or electrostatic contacts (solid edges) primarily with bases in the major groove of the DNA. See embedded legends for detailed key. Diagrams generated using DNAproDB

Fig. 6

DNA Shape and E2 Interaction Analysis for HPV E2-DNA Complexes. The figure displays analyses for (A) the HPV18 E2-DNA complex (PDB: 1JJ4) and (B) the HPV6 E2-DNA complex (PDB: 2AYB). Top Row (Helical Shape Plots): Plots of DNA minor groove width (black line, in Ångstroms) along the E2 binding site sequence. Overlaid points indicate the secondary structure (helix, strand, or loop; see legend) and position of specific E2 amino acid residues interacting at the DNA interface. Bottom Row (Helical Contact Maps): Protein secondary structural elements (SSEs) from E2 are plotted in helicoidal coordinates, showing their position relative to the DNA helical axis and the major/minor grooves (represented as concentric annuli). Specific E2 SSEs (e.g., HA1, HB1 from the recognition helix) are indicated. Diagrams generated using DNAproDB

Fig. 7

Predicted steric and structural impact of the T309P substitution. Models based on AlphaFold predictions were visualized in PyMOL. The structure of the α-7 helix is shown, with critical DNA interaction elements colored blue and the residue at position 309 colored red. The top panel shows the wild-type threonine residue (T309) in its helical context. The bottom panels show the two possible rotamer conformations of the 309P substitution as predicted by the AlphaFold model, with the predicted region of steric clashes highlighted with red circles. The ‘structural kink’ referenced is a disruption to the backbone geometry identified via DSSP analysis of the AlphaFold model, which reclassified the secondary structure at position 309 from ‘Extended Strand’ to ‘β-Bridge’ and at HIS310 from ‘Extended Strand’ to ‘Coil’ in the mutant. These changes highlight potential disruption of the canonical helical structure, which may impact DNA-binding functionality