Figure 1.
Tetrameric structure of SgrAI.
(A) Dimeric structure of SgrAI bound to primary site DNA (PDB code: 3DVO) [16]. Residues of the swapping domain (1–24) shown in space filling spheres. Residues of the hinge loop (25–30) shown as sticks. Residues 31–339 shown as ribbons, and the bound DNA (black) shown as cartoon. Bound Ca2+ ions shown as black spheres. (B) Tetrameric structure of SgrAI with subunits A, B, G, and H labeled and colored in teal, salmon, slate, and sand, respectively. Each subunit swaps the amino-terminal 24 amino acid residues (shown as space filling spheres) with those of a subunit in an opposing dimer. Residues of the hinge loop (25–30) shown as sticks. Residues 31–339 shown as ribbons, and the bound DNA (black) shown as cartoon. Bound Ca2+ ions shown as black spheres. (C) Ribbon diagram of NgoMIV (PDB code: 1 FIU) (subunits in teal, salmon, slate, and sand) bound to DNA (black) and Mg2+ (black spheres).
Figure 2.
Electron density at swapping hinge loops.
(A) 2Fo-Fc SA omit electron density map at 1 σ for residues 23–31 of subunits B (salmon) and G (slate). Ribbon representation of SgrAI subunits shown in swapped conformation. (B) As in (A) with unswapped conformation.
Table 1.
Diffraction data and structure refinement statistics.
Figure 3.
(A) Positions of 2-fold rotational axes of each dimer in tetramer 1, composed of subunits A, B, G, and H, shown as black lines with a black oval. Subunit colors as in Figure 1A–B. Swapped domains and ordered hinge loops represented by circles and lines with the color of their parent subunit. The hinge loop of subunit A is not ordered. (B) Side view of the tetramer shown in Figure 3A showing the positions of the 2-fold axes. The 2-fold dimeric axis of the upper dimer (subunit G, slate; subunit H, sand; Figure 3A) is 9° from that of the lower dimer (subunit A, teal; subunit B, salmon; Figure 3A) and 10° in tetramer 2.
Figure 4.
(A) Close-up of swapped regions of two subunits in the SgrAI tetramer. Residue Arg 31, responsible for recognition of the outer base pair of the primary site recognition sequence, shown as spheres (subunit B, salmon, blue; subunit G, slate, blue). Active site bound Ca2+ ions shown as black spheres and DNA shown in cartoon representation in black. (B) Close-up of interactions at the swapped segments near Pro 27. (C) Same view as in (B) but using unswapped dimer models.
Table 2.
Equilibrium dissociation constants (KD (nM)) for wild type SgrAI dimer (unless otherwise noted) and DNA sequences at 4°C.
Table 3.
Single turnover DNA cleavage rate constants using 1 µM enzyme.
Figure 5.
Stimulation of HMWS formation by PCP.
Native PAGE of 1 µM wild type or mutant SgrAI with 1 nM 32P labeled primary or secondary site (18-1 or 18-2), and increasing concentrations of unlabeled precleaved primary site (PCP, 10, 30, 60, 100, 200, 300, 400, 500, 600, 1,000 nM). (A) wild type SgrAI, (B) P27W SgrAI, (C) P27G SgrAI.
Figure 6.
Red, SgrAI primary site recognition sequence; blue, deviation from primary site recognition sequence; arrows, sites of cleavage by SgrAI.