STUDIES ON THE DNA BINDING DOMAIN OF HUMAN PAPILLOMAVIRUS 33 E2 PROTEIN

Abstract

Human papillomavirus 33, a high-risk HPV strain, belongs to the "Papillomaviridae" family, mainly responsible for HPV infection and cervical cancer in Asian countries. Till now, more than 200 types of HPV are known to the world. These strains can cause small benign lesions or warts to hyperproliferative lesions, which lead to cancers like anal, throat, mouth, cervical, etc. HPV 33 is a small, nonenveloped virus with a diameter of approximately 55 nm. Its genome consists of three main regions: The first region, termed the Long Control Region (LCR), is a noncoding upstream regulatory region housing the p97 core promoter, enhancer, and silencer sequences. The second region, an early region, comprises various Open Reading Frames (ORFs), including E1, E2, E4, E5, E6, and E7, which are crucial for viral replication and oncogenesis. The third region, a late region, encodes the structural proteins L1 and L2, forming the viral capsid. The E2 protein, existing as a homodimer, is about 360 amino acids long with a molecular mass close to 42 kDa. It consists of a conserved N-terminal domain known as transactivation domain (TAD)", size is about 200 amino acid that linked to a C-terminal domain known as DNA binding/dimerization domain (DBD)", size is around 80-100 amino acid. A flexible linker sequence connects the two domains called the "hinge region," which varies in length and amino acid sequence. The E2 DBD is a sequence-specific domain that forms a soluble and stable dimer. It binds to specific consensus motifs, such as ACCGN4CGGT or ACCN6GGT, located mainly in the Upstream Regulatory Region (URR). DNA binding to the E2 DBD governs various essential processes in the viral life cycle. According to the solved structures of E2 DBD, the surface α-helices formed the DNA recognition helix and binds to consensus bases in DNA. In addition to its DNA binding function, the E2 DBD serves a regulatory role and participates in various essential processes such as SUMO modifications. It acts as a site for interacting with the E1 protein and engages with cellular and host proteins like Brd4, p53, PARP, and BRCA1, among others, for transcriptional regulatory functions. Different sites within the E2 DBD mediate these interactions. Several regulatory proteins of HPV are potential targets for vaccine development. However, a permanent cure remains elusive due to the challenge of avoiding adverse effects on untransformed or healthy cells when targeting these proteins. Despite being classified as a "high-risk" type, HPV 33 and its proteins have received limited research attention to date. The HPV 33 E2 DBD emerges as a promising target for cancer prevention, given its pivotal role in critical processes such as replication and transcription. The multifunctional nature of E2 DBD underscores its significance as a key research target. Understanding its structure and binding properties holds promise for identifying and targeting potential interaction sites to mitigate cancer risks. In addition to protein characterization studies, the binding of natural compounds with proteins as inhibitors is well known and plays a pivotal role in regulating the activity of various biological processes in cancer and other disease-related progression. Among these small molecules, polyphenols, naturally found in dietary sources, exhibit potential inhibitory effects on crucial biological processes. Resveratrol and baicalein have many functions, such as anti-inflammatory, antimicrobial, antiviral, and anticancer. Their interaction studies with macromolecules can provide helpful insights into the binding mode and other therapeutic functions. Thus, the present thesis comprehensively details cloning, over-expression, purification, and initial structural and binding characterization of the HPV 33 E2 DBD using biophysical and in silico techniques. This work endeavor represents a significant advancement in this field, as it not only provides foundation knowledge of the HPV 33 E2 DBD but also serves as a lead for future investigations into the other homologous proteins within the HPV family. The thesis is divided into six chapters. Chapter 1 comprises a general introduction and comprehensive literature review of Human papillomavirus and its proteins. It briefly reviews HPV 33, its proteins (E1, E2, E4. E5, E6, E7, L1 and L2), and more specifically E2 DNA binding domain (DBD). The chapter reviews structural and functional information on the E2 DBD protein of different HPV types, polyphenols used (resveratrol and baicalein), structures, biological properties, and binding studies with HPV proteins. Chapter 2 includes the experimental details and methodologies used to reach the objectives. This chapter describes the materials, experimental setups, methods, and equipment used. Chapter 3 presents the cloning, over-expression, purification, and initial characterization studies of HPV 33 E2 DBD using multi-spectroscopic techniques and in silico methods. The preliminary characterization encompassing biophysical experimentation and bioinformatics analysis affirmed its conserved properties compared to other high-risk HPV strains. Notably, our study underscored the soluble nature of the protein in its native state, which is studied by gel exclusion chromatography and exists predominantly as a stable dimer at pH 7-8. The GdHCl-induced denaturation experiment suggested that the process is a non-two-state transition with a transient intermediate-state formation. The study also hints that the protein'sdissociation and denaturation are simultaneous. The in silico investigations indicated that the HPV 33 E2 DBD retains the conservative structural features with its homologous proteins of other strains. The predicted modeled structure of E2 DBD and Ramachandran plot analysis suggested that the modeled structure is of good quality and reliable for further bioinformatic studies. Chapter 4 presents the DNA binding activity of the E2 DBD with the two different lengths of DNA sequences, D12A (12 bp) and D18A (18 bp). Biophysical and in silico experiments suggested the binding interaction of the E2 DBD with both DNA sequences. Both DNA sequences show good binding affinities in the range of 107 M-1, which aligns with the previously reported findings of protein-DNA interactions in the 105 to 108 M-1 range. The Circular dichroism (CD) results infer that no significant conformational changes occur in the E2 DBD native structure upon binding to DNA sequences. The DNA binding impacts the arrangement of the secondary structural elements with little changes in the overall tertiary structure. The Differential scanning calorimetry (DSC) analysis showed the higher thermal stability of E2 DBD-DNA complexes. The melting temperature of E2 DBD (Tm=52.59℃) increased by ΔTm = ̴ 16-20 ℃ upon complex formation with D12A and D18A. The docking and molecular dynamic (MD) simulation studies suggest that both DNA bind to the recognition helix of the E2 DBD and form stable complexes. Hydrogen bonding plays an important role in the stabilization of the complexes. Chapter 5 discussed the binding studies of the natural polyphenolic compounds, resveratrol, and baicalein, renowned for their potential therapeutic properties, with the HPV 33 E2 DBD. Fluorescence investigations implied that resveratrol and baicalein interact with the E2 DBD, leading to fluorescence static quenching with binding constants 1.69 ×107 M-1 and 2.95 ×107 M-1. The CD results suggested that resveratrol and baicalein bound to different sites on E2 DBD. Resveratrol binding brought significant changes at both CD peaks (~210 nm and ~225 nm) of E2 DBD. Meanwhile, baicalein binding did not cause significant perturbation in CD peaks. Further, the DSC analysis showed a high Tm value for both complexes, ΔTm = ̴ 7-12 ℃, confirming both complexes' thermal stability. The isothermal titration calorimetry (ITC) findings suggested that the binding reactions of both polyphenols to E2 DBD were exothermic and had Kd values in the micromolar range. Molecular docking results reveal that resveratrol and baicalein bind to the α-helix and dimeric interface, respectively, with binding affinities of -6.31 kcal/mol and -7.46 kcal/mol. Molecular dynamics simulations and MMPBSA analysis affirmed that throughout the simulation time of 100 ns, the E2 DBD accommodates resveratrol and baicalein at the binding sites, forming stable complexes with minute backbone fluctuations compared to E2 DBD alone. The stability was slightly higher for baicalein than resveratrol, attributed to the unique binding sites and the greater number of hydroxyl groups present in baicalein, enhancing its interaction with E2 DBD. Chapter 6 describes the structure-based virtual screening and docking studies of HPV 33 E2 DBD, a key target in the HPV life cycle and its pathogenesis. This chapter aimed to find potential compounds for E2 DBD protein. The virtual screening resulted in four FDA-approved compounds (ZINC12503187, ZINC4175630, ZINC27990463, and ZINC3817234) with the highest binding affinities that can be used as inhibitors. The molecular docking and simulation studies showed that the selected four compounds showed good binding affinity for E2 DBD from -6.53 kcal/mol to -8.76 kcal/mol. The molecular dynamic studies revealed that the selected ligands bind effectively with the E2 DBD with high stability. The present study gives an understanding of the structural, functional, and interactional properties of the E2 DBD protein, which is crucial to unraveling the knowledge of HPV and its associated molecular mechanisms and pathologies. This will broaden the knowledge of the structural, binding, and functional properties of E2 DBD with DNA and ligands. Targeted drug therapy and designing inhibitors against the E2 DBD dimerization or inhibiting its functions will be the new dimension of this study.