A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

Abstract

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.

Figure 1.

Alignment of the amino-acid sequence of the N-terminal domain of DnaI from B. subtilis with homologous N-terminal regions of different bacterial helicase loaders. The following sequences are shown (organism, abbreviation, GenBank number): B. subtilis, Bsu, NP_390776; B. amyloliquefaciens, Bam, YP_001422191; B. licheniformis Bli, YP_092605; B. pumilus, Bpu, YP_001487763; Geobacillus sp., Gsp, ZP_02915066; Geobacillus kaustophilus, Gka, YP_148574; Geobacillus thermodenitrificans, Gth, YP_001126735; B. species SG-1, Bsp, ZP_01861306; B. cereus subsp. cytotoxis, Bcs, YP_001376478; B. thuringiensis serovar israelensis, Bts, ZP_00742835; B. halodurans, Bha, NP_244010; B. clausii, Bcl, YP_176196; B. selenitireducens Bse, ZP_02170630; Listeria welshimeri serovar, Lws, YP_849770; Lactobacillus helveticus, Lhe, YP_001577829; L. monocytogenes, Lmo, ZP_02289641; Enterococcus faecium, Efa, ZP_00604060; S. haemolyticus, Sha, YP_253156 and S. aureus, Sta, NP_646444. Residues 1–106 of B. subtilis DnaI correspond to the DnaI-N106 domain for which the 3D structure was determined by NMR spectroscopy. An extended version comprising residues 1–123 (DnaI-N123) was also investigated in the present study. The alignment includes sequences with high homology to B. subtilis DnaI-N106 as identified in a BLAST (47) search. The residue numbers and regular secondary structure elements of B. subtilis DnaI-N106 are indicated at the top. Identically conserved residues are highlighted in black. Residues with <10% side-chain solvent accessibility in the 3D structure are highlighted in blue. Four asterisks at the bottom identify the zinc-binding residues of B. subtilis DnaI.

Figure 2.

¹⁵N-HSQC spectrum of B. subtilis DnaI-N106. The spectrum was recorded at 25°C with a 0.5 mM solution of uniformly ¹⁵N-labeled DnaI-N106 at pH 7.0. The cross-peaks are assigned using one-letter amino-acid symbols and residue numbers. Side-chain resonances are marked by small characters and cross-peaks belonging to the same NH₂ group are connected by horizontal lines.

Figure 3.

Solution structure of B. subtilis DnaI-N106. Only residues 15–106 are shown since the initial 14 residues were disordered. The zinc atom is shown as a sphere (magenta) and the side chains of coordinating residues Cys67, Cys70, His84 and Cys101 are shown in yellow (cysteine residues) and blue (histidine). (A) Ribbon representation of DnaI-N106. The secondary structure elements are labeled as in Figure 1. Helix 5 is a 3₁₀ helix found in most but not all of the NMR conformers. (B) Same as (A), but color coded to reflect the changes in backbone amide chemical shifts observed in DnaI-N106 versus those in DnaI-N123 (blue: very small chemical shift changes >0.015 ppm (¹H) or >0.15 ppm (¹⁵N); red: significant chemical shift changes >0.05 ppm (¹H) or >0.5 ppm (¹⁵N). (C) Stereo view of a superposition of the backbone atoms of 20 NMR conformers. (D) Stereo view of a heavy-atom representation of the conformer closest to the mean structure of DnaI-N (only residues 15–106 are shown). The side-chains are color coded in blue (Lys, Arg, His), red (Asp, Glu), yellow (Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Val) and gray (Asn, Gln, Ser, Thr, Tyr). The figure was prepared using the program Molmol (48).

Figure 4.

Solvent accessibility of the amino-acid side chains in DnaI-N106. The solvent accessibilities are expressed as a percentage of their accessibilities calculated for the situation where the respective amino-acid residues are located in a hypothetical poly-Gly α-helix with a fully extended side chain (49). The values were averaged over the 20 NMR conformers. The locations of the helices and β-strands are indicated at the top.

Figure 5.

Binding isotherms for the interaction of Bst DnaB with DnaI-N domain constructs, as measured by SPR. Experimental points obtained with: DnaI-N132 (filled circle), DnaI-N123 (filled square) and DnaI-N106 (filled triangle). The K_D values for these interactions, determined by the least squares fit to a 1:1 binding model (solid lines) are: DnaI-N132, 0.65 μM; DnaI-N123, 7.3 μM; DnaI-N106, 18.5 μM. Given the difference in the K_D values, different ranges of protein concentrations were used: DnaI-N132, 0.1–1 μM; DnaI-N123, 0.1–20 μM; DnaI-N106, 0.1–60 mM. The responses (R, in RU) were normalized using the computed value of the maximum response at equilibrium (R_max), which corresponded to 0.8, 1.7 and 1.8 molecules of DnaI-N132, DnaI-N123 and DnaI-N106, respectively, bound per Bst DnaB subunit.