E1 Helicase
Created by Mohammad Imaad
The Papillomaviral E1 Helicase protein (PDB ID: 2V9P) is a member of the helicase superfamily III of ATPases (7). Its N-terminal half consists of a regulatory domain and a sequence-specific DNA-binding domain involved in the recognition of the replication origin, while the C-terminal half can function alone as a helicase.(1) Helicases couple the energy of nucleotide hydrolysis to the unwinding of double-stranded nucleic acids (3). E1 also performs the initiation function required for helicase loading, generating the single-stranded origin (ori) DNA from which processive unwinding can proceed, melting the origin DNA where they then assemble as helicases.(3)
Helicases couple the energy of nucleotide hydrolysis to the unwinding of double-stranded nucleic acids (3). The helicase domain of the protein can be subdivided into two portions: the oligomerization domain and AAA+ domain containing functional sites involved in ATP hydrolysis and ssDNA translocation(1).
The E1 protein assembles in a stepwise fashion on the ori (3). The initial binding of E1 as a dimer requires the assistance of the transcription factor E2, which orients subsequent oligomeric complexes in a head to tail array(3). Two E1 trimers melt the DNA on either side of the E1-binding site, a process, which is ATP dependent and requires the cooperation of the ssDNA-binding site in the E1 Helicase molecule (2,3).
Papillomaviruses are tumor viruses within a host cell and require the multifunctional 605-residue viral E1 protein (1,2). Monomers of E1 assemble at the replication origin on DNA and form a pair of hexamers that wrap around a single strand of DNA(1). These assemblies are helicases that operate bidirectionally in the replication of viral DNA (1,2).
E1 protein's N-terminal half (residues 1-300) consists of a regulatory domain and a sequence-specific DNA-binding domain involved in the recognition of the replication origin, while the C-terminal half can function alone as a helicase (2,7). The helicase domain of the protein can be subdivided into two portions:the oligomerization domain (residues 300-378) and AAA+ domain (residues 378-605), containing functional sites involved in ATP hydrolysis and ssDNA translocation(7).
AAA+ domains consist of two subdomains: an N-terminal segment called the alpha/beta Rossmanfold, and a C-terminal a-helicaldomain.(7,2). The Rossman fold is wedge-shaped and has a b-sheet of parallel strands in a b5-b1-b4-b3-b2 pattern (7). Key features of this fold include a lysine residue in the Walker A motif (GPPNTGKS), an aspartate-aspartate pair in theWalker B motif (AALVDD), and a crucial arginine residue in an arginine finger (7). These three motifs are essential for ATP binding and hydrolysis (1,2,5).
Secondary Structure of E1 Helicase
In an AAA+ hexamer, the ATP binding sites lie at the interface between any two subunits, involving the arginine finger of any given subunit and the Walker A and Walker B motifs of an adjacent subunit (1).
The structure of a large fragment (residues 306-577) of the papillomavirus E1 protein bound to a segment of ssDNA allows for the molecule to traverse a DNA chain(7). These allow for the 2 confirmations of the E1 Helicase: the simple E1 Helicase (2V9P), andE1 Helicase bound with ssDNA and MgADP (2GXA) (7). The oligomerization domains form a symmetric hexamer, but the six AAA+ domains each display a unique conformation(7). The DNA strand is bound in the center pore of the AAA+ hexamer with six nucleotides of the DNA chain eachbound to the residues from each of the protein subunits(7). Here crucial nucleotide binding residues work, where Lys506 interacts with one DNA phosphate oxygen, and His507 forms a hydrogen bond with the phosphate of an adjacent nucleotide in the DNA chain (1).
There are many similarities between E1 Helicase and SV40 large T antigen helicase (PDB ID: 1SVM) in terms of structure and function. These can be expressed after research and the DALI results(Z score: 36.7) observed showing a similarity in helicase activity observed in both proteins. (8) The large T antigen helicase is a hexameric helicase essential for viral DNA replication in eukaryotic cells. LTag functions as an efficient molecular machine powered by ATP binding and hydrolysis for origin DNA melting and replication fork unwinding (3,8,6).
The aliphatic portion of Lys506 and the aromatic groups of Phe464 and His507 share van der Waals interactions with the DNA sugar moiety linking the two phosphates (1).
The hairpin loops of the six protein subunits for a spiral staircase, following the ssDNA as it threads through the central pore of the hexamer (7). Then as each AAA+ domain proceeds through the intermediate states of ATP binding and hydrolysis, its hairpin loop steps down through the six confirmations of the staircase maintaining contact with the nucleotide, releasing it as it exits the pore, then moves back to the top to pick up the next available nucleotide (7). A full cycle translocates six nucleotides with associated hydrolysis of six ATPs and release of six ADPs (7).
All of these subunits correspond to two phosphate ions and one magnesium ion (1,4).
Analysis of the six ATP-binding sites of the DNA-nucleotide-free E1 hexamer shows they exist in three different states, ATP-type, ADP-type and apo-type (1,2). The configuration ofthree active sites formed by subunits A/B, B/C and C/D, with two bound phosphates and a magnesium ion, corresponds to the ATP-type of nucleotidecoordination (1). Active sites formed by subunits D/E and E/F with onebound phosphate ion assume the ADP-type coordination, where Y534 is sandwiched between R493 and R538 (2). E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA(ssDNA) or both(2).
In most cases ssDNA is the preferred substrate for DNA binding, and during its presence, oligomerization is highly cooperative(2).
Scientists recently described novel biphenylsulfonacetic acid inhibitors of the ATPaseactivity of E1 from HPV6 (3). Studies implicate Tyr-487 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts. Besides these studies there are no other drugs targeting or intereacting with the protein discovered as of yet. (3)