The hexameric helicase DnaB adopts a nonplanar conformation during translocation - PubMed (original) (raw)

The hexameric helicase DnaB adopts a nonplanar conformation during translocation

Ornchuma Itsathitphaisarn et al. Cell. 2012.

Abstract

DNA polymerases can only synthesize nascent DNA from single-stranded DNA (ssDNA) templates. In bacteria, the unwinding of parental duplex DNA is carried out by the replicative DNA helicase (DnaB) that couples NTP hydrolysis to 5' to 3' translocation. The crystal structure of the DnaB hexamer in complex with GDP-AlF(4) and ssDNA reported here reveals that DnaB adopts a closed spiral staircase quaternary structure around an A-form ssDNA with each C-terminal domain coordinating two nucleotides of ssDNA. The structure not only provides structural insights into the translocation mechanism of superfamily IV helicases but also suggests that members of this superfamily employ a translocation mechanism that is distinct from other helicase superfamilies. We propose a hand-over-hand mechanism in which sequential hydrolysis of NTP causes a sequential 5' to 3' movement of the subunits along the helical axis of the staircase, resulting in the unwinding of two nucleotides per subunit.

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Figures

Figure 1. Overall architecture of the cocrystal structure DnaB6 with ssDNA and GDP-AlF4

A) Domain organization of a DnaB monomer B) Initial experimental electron density at 5.9 Å resolution contoured at 2σ (green) and final 2Fo-Fc electron density at 3.55 Å resolution contoured at 2σ (blue) C) A right-handed lockwasher D) Top view of DnaB6. The N-terminal domain (NTD) trimer-of-dimers and linker domains are shown in cartoon representation, while the C-terminal domain (CTD) staircase is shown in solvent accessible surface representation. The ssDNA and five molecules of GDP-AlF4 are shown as yellow and black spheres respectively. E) Side view of DnaB6 rotated 90° with respect to (D). The C-linkers that are not visible in the electron density map is modeled as blue dashed lines. See also Figure S7

Figure 2. Interactions between an A-form ssDNA and the DNA-binding loops in the CTD staircase

A) The DNA-binding loops of DnaB6 form a right-handed spiral staircase along the A-form ssDNA with a pitch of 32 Å, which is depicted in yellow cartoon representation. The CTD subunits are shown as cartoon with the residues involved in DNA-binding displayed as sticks. B) The spiral staircase of nucleic acid-binding loops of Rho (PDB ID: 3ICE) and E1 (PDB ID: 2GXA). One of the RNA binding loops of Rho is removed for clarity. Subunits are colored according to their positions along the spiral staircase as in (A). Each helicase-bound nucleic acid substrate is displayed as yellow cartoon with its pitch shown on the left. C) The helicase-ssDNA interactions of the boxed region in (A) and representative Fo-Fc electron density for DNA. The Fo-Fc density that was calculated prior to including the ssDNA in the model is shown in orange and black mesh at contour levels 2σ and 3σ, respectively. For a pair of CTD subunits, the CTD5′ is the subunit that interacts with the nucleotides at the 5′ end of the ssDNA and is equivalent to an upper step of the staircase, while CTD3′ is the subunit that contacts the nucleotides at the 3′ end of the ssDNA and acts as a lower step in the staircase. The phosphate backbone of the ssDNA is recognized by the amide groups in the peptide backbone of E382 and E384 as well as the sidechain of R381. D) The hydrogen bond networks that connect adjacent DNA-binding loops and link the active site to the DNA-binding loop. See also Figure S2

Figure 3. Formation of a NTP-bound active site

A) Detailed interactions between the active site residues and GDP-AlF4 in a representative active site (between subunits E and D). Representative Fo-Fc electron density of GDP-AlF4 in the NTPase active site calculated prior to including GDP-AlF4-Ca2+ in the model is shown in green mesh at the contour level of 2σ. Nucleotide binding residues from the CTD5′ include Walker A-motif (WA: G215, K216, T217), Walker B motif (WB: D320), a catalytic glutamate (CE: E241), R250 and Q362. R250 coordinates the α-phosphate. Nucleotide binding residues from the CTD3′ include K418 and R420. See Figure S1 for final 2Fo-Fc electron density of GDP-AlF4 and the active site residues. B) Subunit rotation upon GDP-AlF4 and ssDNA binding. The diagram was generated by superimposing one subunit from the unliganded DnaB6 structure (PDB ID: 2R6A) on the subunit of the current DnaB6 structure that provides the Walker A motif for GDP-AlF4 (CTD5′) by a least square method. Only the adjacent subunits that donate the Arg-finger for NTP coordination (CTD3′) are shown, while the subunits used in the superimposition are omitted. The dark pink subunit is from the DnaB6-ssDNA-GDP-AlF4 complex, while the blue subunit comes from the unliganded DnaB6. DNA-binding loops and Arg-fingers are shown as spheres, while DNA is shown as a yellow cartoon. GDP is represented with green sticks.

Figure 4. The hand-over-hand mechanism of DnaB6

Subunits are colored as in Figure 1. The lagging strand on which DnaB6 translocates is colored yellow while the leading strand is colored orange. Red nucleotides illustrate residues that interact with the helicase in the crystal structure presented here, while green nucleotides represent those that are entering the hexamer after ATP turnover. The thick black arrow denotes the downward movement of the top CTD subunit towards the 3′ end of the lagging strand. See also Figures S3, S4, S5 and S6 and Movie S1

Figure 5. Synchronization of the NTD staircase formation with the NTP hydrolysis cycle in the CTD staircase

A) Top view down the 5′ end of the ssDNA substrate. The NTD (top) and CTD (bottom) staircases are shown as dimers and circles that are spiraling into the plane of the paper, respectively. The sizes of the subunits indicate the positions of the subunits along the spiral staircase with the largest symbols representing the top subunits and the smallest symbols representing the bottom subunits. Black triangles denote sites where the NTD and CTD staircases split into the lockwasher. In the CTD panel, the black triangles also illustrate the binding site of incoming NTP while white triangles indicate the site of NTP hydrolysis. B) The NTD trimer-of-dimers would act as a 3-step staircase that splits after the translocation over 4 base pairs whereas the CTD staircase splits after two basepairs. State 1 is the current structure, while states 3 and 5 are generated by 120° and 240° clockwise rotation of state 1 along the ssDNA axis. See also Movie S2

Comment in

A helicase with an extra spring in its step.
Schlierf M, Ha T. Schlierf M, et al. Cell. 2012 Oct 12;151(2):244-6. doi: 10.1016/j.cell.2012.09.031. Cell. 2012. PMID: 23063116

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The hexameric helicase DnaB adopts a nonplanar conformation during translocation - PubMed (original) (raw)