Crawling and Wiggling on DNA Kenneth J Marians  Structure  Volume 8, Issue 12, Pages R227-R235 (December 2000) DOI: 10.1016/S0969-2126(00)00539-6

Figure 1 The Product and Substrate Complexes of PcrA Bound to Forked DNA (a) The product complex and (b) the substrate complex of PcrA. Domains 1A, 2A, 1B and 2B are colored green, red, yellow and blue, respectively. The sulfate ion in (a) and the ADPNP molecule in (b) are shown in gold, and the DNA is in magenta. (The figure was reproduced from [6] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 2 Cleft Closure Triggered by Binding of ADPNP to PcrA (a) Cleft open and (b) closed as seen from the typical side view. (The figure was reproduced from the Wigley lab web site with the kind permission of D. Wigley and M. Dillingham.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 3 Disposition of the Duplex DNA when Bound to PcrA The binding surface is colored to indicate electrostatic surface potential, blue is positive and red negative. Separation of the duplex strands at the junction is obvious, as are the different paths of the two strands along the surface of the protein once separated. On the left, the thymidine tail — the strand that the enzyme is “bound” to — runs back into the groove that is formed between domains 1A and 2A, whereas the other strand angles off in a completely different direction. (The figure was reproduced from [6] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 4 A Model for ssDNA Translocation (a) Location of the aromatic residues that form the DNA binding pockets across the surface of domains 1A and 2A in the substrate complex. The domains are colored as in Figure 1. The six aromatic amino acid residues that form the binding pockets are colored magenta and are, from left to right, Phe626, His587, Trp259, Tyr257, Phe64 and Phe192. Mutations in these residues have pronounced affects on the PcrA DNA helicase activity (D. Wigley, personal communication). This figure was prepared by M. Dillingham using the program RIBBONS [30] and is reproduced with his permission. Disposition of the ssDNA in the binding pockets in (b) the product complex and (c) the substrate complex. There are three bases in equivalent positions in the two complexes, but they are displaced by one nucleotide residue. (d) The relative movements of domains 1A and 2A as the protein binds and hydrolyzes ATP. An open hand represents a “loose” grip on the ssDNA whereas a closed fist represents a “tight” grip on the ssDNA. (e) Relative movements that occur as the bases flip in and out of binding pockets during translocation along ssDNA. Base numbering is in the 3′ → 5′ direction. (Parts [b]–[e] were reproduced from [6] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 5 Structure of the Hexameric Gene 4 Protein Helicase Domain (a) The helical filament formed in the crystallographic unit cell by six molecules (each colored differently) of residues 272–571 of the helicase domain viewed perpendicular to the 61 screw axis (shown as a black line). dTTP is shown as van der Waals spheres. (b) The ring-like appearance of the six molecules shown in (a) when viewed parallel to the 61 crystallographic axis. The conserved helicase motifs are in red (H1), yellow (H1a), green (H2), cyan (H3), and purple (H4). The black loops in the center represent the disordered DNA binding loops. (Parts [a] and [b] were reproduced from [7] with permission.) (c) The monomer unit of the hexameric crystal formed by residues 241–566 of the helicase domain. Colored from blue at the N terminus to red at the C terminus. ADPNP is shown in ball-and-stick representation. (d) Adjacent monomers in the hexamer interact via the N-terminal region (residues 264–284) of one subunit and a pocket (residues 364–395) of its neighbor. ADPNP is shown in ball-and-stick representation. The view is from the N-terminal side of the ring. (The figure was reproduced from [8] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 6 Asymmetry of the Hexameric Ring (a) The position of the gold atoms in a heavy-metal derivative show the asymmetry of the ring about both a side and top view. (The figure was reproduced from the Wigley lab web site with the kind permission of D. Wigley and M. Singleton.) The distances between gold atoms in pairs of subunits are indicated in angstroms. (b) The asymmetry in the ring results in the DNA binding loops, colored green, red and blue, spiraling across the inner surface of the central cavity. A, B, and C refer to the relative rotations of the monomer units as described in the text. This cutaway view is from the inside of the ring looking out and the C-terminal face of the ring is on top. The dotted line is the symmetry axis for the hexamer. (The figure was reproduced from [8] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 7 A Binding Change Mechanism for NTP Hydrolysis by the g4P Helicase Because the B sites shows the highest occupancy for ADPNP it is assumed that they bind ATP. The A sites, which have a lower occupancy, would bind ADP and Pi. The C sites are empty. Hydrolysis of ATP at the B sites causes the propagation of a conformational change around the ring that allows the C sites to bind ATP and the A sites to discharge their hydrolysis products. Repetition of the cycle allows each subunit to hydrolyze ATP, discharge the products of hydrolysis, and become empty in turn. Thus, as opposed to the F1-ATPase where the γ subunit actually rotates [31], in the g4P helicase ATP hydrolysis undulates around the ring. (The figure was reproduced from [8] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)

Figure 8 Model for Translocation Along ssDNA by the g4P Helicase (a) The relative movements of the DNA binding loops in three consecutive subunits as the ATPase-driven conformational changes migrate around the ring. (The figure was reproduced from the Wigley lab web site with the kind permission of D. Wigley and M. Singleton.) (b) Translocation as viewed from inside the ring looking out at the subunits that have been flattened into two dimensions. The arrangement of the subunits is as shown in Figure 6. The DNA binding loops are in red and the ssDNA is alternately colored in orange and black with each segment representing the translocation step size. (c) Translocation as viewed from outside the ring. The step segments are alternately colored as in (b) but the segment that contacts the protein is in cyan. The front three subunits of the ring are transparent to allow viewing. The DNA tracks around the inside of the ring with neither molecule rotating significantly. (Parts [b] and [c] were reproduced from [8] with permission.) Structure 2000 8, R227-R235DOI: (10.1016/S0969-2126(00)00539-6)