Volume 13, Issue 4, Pages 609-616 (April 2005) Solution Structure of the Helicase-Interaction Domain of the Primase DnaG  Karl Syson, Jenny Thirlway, Andrea M. Hounslow, Panos Soultanas, Jonathan P. Waltho  Structure  Volume 13, Issue 4, Pages 609-616 (April 2005) DOI: 10.1016/j.str.2005.01.022 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Helicase-Interaction Domains of DnaG Primary sequence alignment of the helicase-interaction domain of DnaG from (top down) Bacillus stearothermophilus, B. subtilis, B. halodurans, B. cereus, B. anthracis, Oceanobacillus iheyensis, Lactobacillus plantarum, Listeria innocua, and L. monocytogenes, according to the ProDom database (Servant et al., 2002). The equivalent sequence from Escherichia coli (bottom) was aligned using CLUSTALW, carried out with the full-length proteins. The residue numbering and the distribution of secondary structure elements (h = residue in a helical conformation) refer to Bacillus stearothermophilus DnaG. Largely conserved residues are colored green (hydrophobic), orange (acidic), cyan (basic), or yellow (Ala/Gly or Ser/Thr). Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Solution Structure of the Helicase-Interaction Domain P16 Superimposition of 10 representative structures overlaid on the helical elements of the C1 subdomain (A) and of the C2 subdomain (B). The six helices of the C1 subdomain correspond to residues 459-472, 475-484, 492-506, 513-517, 523-532, and 542-553, and the two helices of the C2 subdomain comprise residues 558-574, and 580-594. (C) Cartoon representation of the secondary structure elements in P16. In the C1 subdomain, the first two helices are colored yellow, the middle two are green, and the final two are cyan. The two helices of the C2 subdomain are colored blue, except the region where a slow helix-coil transition occurs, which is colored orange. The loops where rapid solvent exchange of amide hydrogens occurs are colored red. (D) Cartoon representation of the structure of the N-terminal domain of DnaB from E. coli (Fass et al., 1999), orientated using DALI and colored according to the representation of the C1 domain of P16 shown in (B). Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Mobility within the Helicase-Interaction Domain P16 Transverse relaxation times of backbone 15N nuclei within the helicase-interaction domain, in comparison with the distribution of helices (represented by open boxes). Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Electrostatic Surface Potentials Surface charge distribution of (A) the C1 subdomain of P16, (B) the C2 subdomain of P16, and (C) a model of the N-terminal domain of DnaB from B. stearothermophilus, based on the structure of this domain from E. coli. The secondary structure elements are oriented similarly within the left-hand series and within the right-hand series. The right-hand series are 180° rotations of the left-hand series about a vertical axis in the plane of the page. Areas of positive and negative charge on the surface are colored blue and red, respectively. Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 The 3HF Peptide Binds to DnaB but Does Not Stimulate Its ATPase Activity (A) Left gel: Purified 3HF was immobilized onto a HisTrapTM Ni2+-loaded column and DnaB was loaded onto the column (see Experimental Procedures). Fractions from the flow-through during DnaB loading (lane 1) and wash steps with 20% elution buffer (lane 2) and 100% elution buffer (lanes 3 and 4) were analyzed by SDS-PAGE. DnaB and 3HF proteins eluted together in the same fractions, as indicated. Lane M shows molecular weight markers. Right gel: The equivalent experiment without the addition of 3HF. (B) A bar chart showing the effect of 3HF on the ATPase activity of DnaB alone and in complex with DnaG, as indicated. BSA was used as a negative control. The ATPase rate is defined as molecules of ATP hydrolysed per second. Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 6 A Model for the DnaB-P16 Interaction (A) The 3-fold symmetric ring of hexameric DnaB, showing the N-domain of one monomer (6N) interacting with the C-domain (5H) of the neighboring monomer (Yang et al., 2003). (B) In the DnaB-P16 complex, the P16 protein (shaded purple) interacts with the linker region that connects the N-terminal (6N) and C-terminal (6H) domains of one monomer, via its C2 subdomain. In addition, the C1 subdomain displaces 6N while at the same time maintaining the interactions with 5H that are essential to preserve a 3-fold symmetric ring in the helicase-primase complex. Structure 2005 13, 609-616DOI: (10.1016/j.str.2005.01.022) Copyright © 2005 Elsevier Ltd Terms and Conditions