Helicase
Created by Evans Wralstad
The replicative helicase of Enterobacteria phage T7 posesses a gene 4 protein (gp4) essential to DNA replication: the dual DNA primase-helicase protein (1). The C-terminal end of this protein is the helicase domain and is fully functional following proteolytic cleavage from the DNA primase-helicase protein (2). Helicase proteins initiate DNA replication by unwinding duplex DNA to be made accessible to other proteins of the replisome- the supramolecular array of proteins essential to DNA replication (1,3). All helicases are dependent on nucleoside triphosphates (NTPs) and bind to one strand of a duplex DNA segment (3).
Association of a helicase with a larger protein is unusual, as helicases are often solitary proteins. This coupling of functions in gp4 ensures that DNA primase may immediately catalyze primer formation at the replication fork following single stranded DNA (ssDNA) separation by helicase; this is an improvement from other organisms, where homologous helicase proteins form a non-bonded complex with primase proteins to expedite DNA replication. For phage T7 a tight association forms between proteins of the replisome as the DNA primase-helicase protein coalesces with gp2.5 ssDNA binding protein, gp5 DNA polymerase catalytic subunit, and E. coli processivity factor thioredoxin (1).
Phage T7 helicase assumes a hexameric structure as a closed ring in the presence of dNTP, even when cleaved from the primase domain. The crystal structure of the helicase domain consists of a six-subunit helical turn unit cell; however, collapsing this helix along the major axis results in the topologically closed ring observed by electron microscopic imagery (1, 4). This hexameric ring structure is found in other replicative helicases, including that of Bacillus phage SPP1 (PDB ID = 3BGW) (side view,top view) and Geobacillus kaustophilus HTA426 (PDB ID = 2VYE) (side view, top view) (5, 6). The prevalence of this structure indicates the high degree of conservation found among helicase proteins.
The formation of a ring structure is effective in enclosing ssDNA and forces the two strands of duplex DNA apart by the movement of the topologically closed ring, like a bead moving along one of two intertwined threads. This increases the processivity of the DNA helicase, defined as the extent to which a protein is able to continue its catalytic activity before dissociating from the substrate. The enhanced processivity of helicase allows for expedient replication of DNA. The half-life of gp4A dissociation from single-stranded DNA (ssDNA) is about three minutes, allowing for replication of the entire phage T7 genome before helicase dissociation occurs (7).
Phage T7 helicase subunits have a molecular weight of 32,790.97 Da and a theoretical isoelectric point of 5.08 (8). T7 phage helicase is 39.3% α-helix (10 helices spanning 95 residues), 23.1% β-sheet (1 mixed sheet containing 9 strands, spanning 56 residues), 1.2% 3/10 helix (spanning 3 residues), and 36.4% other (consisting of random coil and disordered structure) (9). Helicase subunits are characterized as globular proteins (see the carbon skeleton and orientation of polar/nonpolar residues in the protein).
Catalytic activity of hexameric gp4A requires several ligands: a deoxynucleoside triphosphate (dNTP) to be hydrolyzed to drive helicase activity, duplex DNA strands to be separated, and a divalent metal cation (most commonly Mg2+) to participate in hydrolysis of the dNTP (2). These characteristics are shared in two comparison proteins identified by BLAST and the Dali server: the aforementioned Bacillus phage SPP1 DnaB-like replicative helicase and the Geobacillus kaustophilus HTA426 replicative DNA helicase (10, 11). Gp4A is homologous to B. phage SPP1 helicase with E = 2e-99 and to G. kaustophilus helicase with E = 4e-95. The similarities between the query sequence and the identified homologous sequences are clearly significant and do not occur by chance. These homologous sequences also share structural similarities, with B. phage SPP1 helicase scoring Z = 24.1 and G. kaustophilus helicase scoring Z = 22.7 by Dali. Several similarities are found between G. kaustophilus helicase and gp4A beyond a common homohexameric architecture. In both proteins an NTPase domain is formed at the interface of two protein subunits with an arginine finger motif extending from the subunit adjacent to the interface (Arg-522 for gp4A, Arg-414 for G. kaustophilus), a structure involved in the hydrolysis of the dNTP (1, 5). Both proteins are also topologically closed to encircle ssDNA and enhance processivity. The form of this ring is not identical between the two homologous proteins. For gp4A the hexameric ring is toroidal, with each subunit forming one-sixth of the total ring (1). In G. kaustophilus the ring is triangular in shape and is framed by three subunits; the other three subunits of the hexamer act as a second frame for the ring, forming dimeric interactions with the three subunits bounding the inner ring (5). These two ring forms are among the most common seen for hexameric helicases (7). Gp4A also constitutes only the C-terminal domain of a bifunctional protein, while G. kaustophilus helicase is a solitary and monofunctional protein.
Two models exist to explain how how duplex DNA is unwound to separate ssDNA: the inchworm model and the active rolling model. Both models acknowledge the H4 motif of gp4A, a primarily helical region at the interior of the hexameric ring that contains a 3/10 helix that binds to DNA by the interactions of Arg-487 and Gly-488, and in both models the processive motion of gp4A is motivated by dNTP hydrolysis (1, 2). The inchworm model suggests a cyclic motion along ssDNA that appears spring-loaded; the translocation along DNA and the separation of duplex strands occurs in discrete steps, not continuously. The active rolling mechanism proposes a continuous movement along ssDNA and the smooth separation of duplex strands to the extent that gp4A processivity allows. Both models have been confirmed for helicases homologous to gp4A (12, 13); thus, the mechanism of unwinding by gp4A cannot be annotated by comparison to homologues. Both models require modification to fit the activity of gp4A, however; in neither model does the architecture of the helicase domain optimally suit the model (1).
The binding and hydrolysis of dNTPs, as well as coupling to DNA unwinding, is much better understood for gp4A, and the NTPase domain of gp4A has been extensively modeled. NTPase activity occurs at the interface of two subunits. This activity requires primary association of the dNTP with several residues of the subunit to which the dNTP is bound and secondary association with a key residue of the adjacent subunit. Arg-504 and Tyr-535 are two highly conserved residues that form a pocket in which the nitrogenous base of the dNTP can stack. These residues are nonspecific and allow gp4A to act on multiple dNTPs. A divalent metal cation is also present near the ligated dNTP and is itself ligated by an ionic interaction with Asp-424 (1). It is suggested that Ser-319 participates in metal ion ligation based on comparison to the homologous protein B. stearothermophilus PcrA, in which a threonine residue corresponding to Ser-319 ligates a magnesium ion (1, 14). The bound metal ion is required for hydrolysis of bound dNTPs and participates by properly orienting the scissile γ-phosphate to be hydrolyzed and stabilizing the partial negative charge that develops in the transition state of hydrolysis. The α- and β-phosphates of the bound dNTP are hydrogen-bonded to Ser-319 and Thr-320, stabilizating and orientating the dNTP. Lys-318 of the dNTP binding region also forms a hydrogen bond with a nonbridging oxygen of the β-phosphate (1). These collective residues are part of the Walker A motif (or phosphate binding loop: P loop) found in many proteins that bind nucleotides. The lysine residue corresponding to Lys-318 is highly conserved in the Walker A motif and is an essential residue for dNTP binding (15).
The hydrolysis of a dNTP requires the action of two crucial residues. The arginine finger motif of one subunit, characterized by Arg-522, makes contact with the scissile phosphate of the neighboring subunit and polarizes the γ-phosphate of the dNTP, facilitating hydrolysis. Glu-343 is the key catalytic residue of dNTP hydrolysis and polarizes a water molecule found within the P loop binding pocket. This polarization facilitates nucleophilic attack on the γ-phosphate to initiate dNTP hydrolysis (16). His-465, making contact with the scissile phosphate via hydrogen bonding, acts as a γ-phosphate sensor. Upon hydrolysis this interaction is lost, resulting in a conformational change that propagates throughout the protein to the DNA binding motif H4 (1). This couples dNTP hydrolysis to DNA binding. The hydrolysis of the dNTP also results in the displacement of Arg-522 into a position where it makes contact with Glu-348 of the adjacent subunit. This results in the repositioning of Phe-523 to a pose in which it can interact with the ssDNA excluded from the closed ring of the hexamer. It is thought that the collective results of dNTP hydrolysis— the altered conformation of the H4 motif, allowing Arg-487 and Gly-488 to bind to DNA, and the altered posr allowing Phe-523 to interact with the excluded strand— rotate the duplex strands relative to each other, facilitating destabilization of the inter-strand hydrogen bonds (see the relative orientations of binding, catalytic, and sensing residues) (2).
The mechanism of these interactions, alongside the high degree of conservation found amongst helicase proteins, allows for the annotation of putative helicase proteins by comparison to the model phage T7 replicative helicase. This is of biological relevance in understanding the structure and function of helicase proteins and the diseases that arise from defects in helicase construction or activity.