Nucleotide and partner-protein control of bacterial replicative helicase structure and function

Abstract

Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors.

Figure 1. AaDnaB structure

A) Domain organization. Specific regions discussed in the text are labeled. B) Cartoon representation of hexameric AaDnaB, shown in three views. Coloring of structural elements as per panel A. C) Composite-omit electron density (at 4σ) of ADP•Mg²⁺ and nearby residues. D) Reference-free 2D class averages of AaDnaB compared to forward re-projections of both the 3D EM model and the crystal structure. E) Fitting of the AaDnaB crystal structure into the 3D EM reconstruction.

Figure 2. The DnaB N-terminal collar is a conformational switch

N-terminal collar (A), and top (B) and side (C) views of the C-terminal motor ring for ADP-bound AaDnaB (left), DNA- and GDP•AlF_x-bound GstDnaB (middle, PDB ID 4ESV), and apo GstDnaB (right, PDB ID 2R6A). Subunits are alternatingly shaded light and dark to emphasize the transitions between N-terminal domain homodimers. The angle of the transducer helix (green) orientation relative to the plane of the ring is shown above the side views of the motor domain (C).

Figure 3. Nucleotide promotes DnaB constriction

A) Reference-free 2D class averages of wild type EcDnaB and two collar mutants with different nucleotides. Dilated EcDnaB and constricted AaDnaB EM models are shown at top for comparison. B) Schematic design of the N-terminal collar mutants; green “S-S” connectors represent disulfide bonds formed in the dilated state, while red “X’s” correspond to amino acids in EcDnaB that, when mutated, prevent formation of the dilated state. C) Experimental SAXS curves of EcDnaB with 1mM AMPPNP (black), constricted EcDnaB (orange), dilated EcDnaB (green) and average between the two states (pink dotted line). Inset: theoretical scattering curves of dilated (green) and constricted (orange) DnaB rings, as well as the average of the two models (pink dash) – curves are offset for clarity.

Figure 4. The N-terminal collar conformation affects helicase interactions with DNA and ATPase rate

Panels (A) and (B) correspond to single-stranded DNA binding and duplex DNA binding by wild type (black), constricted (orange) and dilated (green) EcDnaB, respectively, as measured by fluorescence anisotropy (mA – milli-anisotropy units). Panels (C) and (D) correspond to ATP hydrolysis in the presence of single-stranded DNA (dotted lines in panel C) and duplex DNA (dotted lines in panel D), respectively. E) Schematic outline of experiment assaying translocation along duplex DNA. F) Kinetic data showing the ability of wild type (black), constricted (orange) and dilated (green) EcDnaB to translocate along duplex DNA.

Figure 5. DNA unwinding and DnaB-partner interactions are influenced by collar state

A) Basal unwinding levels of a short forked substrate by wild type EcDnaB (black) and both constricted (orange) and dilated (green) DnaB mutants. B) DnaC-dependent activation of DNA unwinding by DnaB and both constricted and dilated DnaB mutants. DnaC preferentially activates DNA unwinding by DnaB helicases that can access a dilated collar conformation. C) Effect of τ on DNA unwinding by DnaB. τ preferentially activates DNA unwinding by DnaB when the helicase favors a constricted collar conformation. D) Primer synthesis by DnaG in the presence of wild type and collar mutants of EcDnaB. Only helicases that can adopt a dilated collar stimulate primer synthesis.

Figure 6. Potential roles for conformational switching in the N-terminal collar as a means to regulate DnaB function

Schematic illustrating how DnaB could use different collar states to switch from basal, low-activity states into translocation-competent forms, and how both substrates and partner proteins preferentially interact with and/or help control collar transitions and ring opening. Dashed lines represent less favored transitions based on data presented here and from the literature.