We have solved three new structures of Sgr
AI bound to DNA and Ca2+
which add to our understanding of the mechanism of Sgr
AI self-activation. Firstly, two isomorphous structures of Sgr
AI bound to a secondary-site DNA containing a noncanonical G·C base pair in one of the two Y
base-pair positions of the recognition sequence CR
G (with 5′-CA|CCGGG
G-3′ on the top strand and 3′-GT|GGCCC
C-5′ on the bottom) have been determined. The crystals were prepared with either Ca2+
. The two structures are very similar, within coordinate error, to each other as well as to the previously solved structure of Sgr
AI bound to a primary-site DNA and Ca2+
(Dunten et al.
), possessing no large conformational changes, with the exception of the absence of divalent cation binding in the M3 metal ion-binding site. Only the M1 site is occupied in the structures with secondary-site DNA and Mg2+
, as was found in the structures of the endonucleases Hin
cII (Etzkorn & Horton, 2004a
) and Bgl
I (Newman et al.
) bound to their cognate DNA sequences and Ca2+
(Newman et al.
). The asymmetric unit of the structures of Sgr
AI with secondary-site DNA contains a dimer of Sgr
AI bound to a full duplex of DNA, but the asymmetric sequence of the DNA appears to be positioned in both directions in the many copies of this complex in the crystal. Therefore, the electron density is averaged at the site of the substitution and refinement was performed with 50% occupancy of each sequence (GC and TA). Analysis of the residues at and around this position indicates no differences in the conformation of the two sequences (green and pink, Fig. 3) nor any significant differences relative to the structure of Sgr
AI bound to primary-site DNA (yellow, Fig. 3). Lys96, which has been postulated to function in indirect readout of the degenerate base pair at the second and seventh base pair of the recognition sequence, is similarly positioned in the structures and the unstacking of G8 from the base at position 7 is also preserved (Fig. 3).
The common enzyme conformation of Sgr
AI bound to uncleaved primary-site (Dunten et al.
) and secondary-site DNA (current work) supports the proposal that these structures are in the low-activity conformation (Dunten et al.
), since secondary-site DNA alone is incapable of activating Sgr
AI (Park, Stiteler et al.
). The common conformation is consistent with the similar DNA-binding affinities (0.6 and 2.6 nM
for 18 bp DNA containing primary-site and secondary-site sequences, respectively; Park, Stiteler et al.
) and similar basal unstimulated DNA-cleavage rate constants (0.09 and 0.02 min−1
for primary-site and secondary-site DNA, respectively; Park, Stiteler et al.
). However, in the absence of stimulation only a small fraction of the secondary-site DNA is cleaved (10%) and stimulation appears to occur by increasing not so much the rate of cleavage (which is stimulated only two to threefold for secondary-site DNA to 0.05 min−1
, while it is stimulated >200-fold for primary-site DNA to 22 min−1
), but the fraction of the DNA that is cleaved (Park, Stiteler et al.
). Therefore, secondary-site DNA is never cleaved as quickly as primary-site DNA under stimulating conditions, suggesting that the secondary site prevents the enzyme from attaining the fully activated conformation.
The structure of Sgr
AI bound to cleaved primary-site DNA and Mg2+
shows a similar conformation to the structures with uncleaved secondary-site and uncleaved primary-site DNA (Fig. 5
), with differences in conformation only at the cleaved phosphate and the nucleotides immediately 5′ to the cleavage site (Fig. 5
). The low activity of Sgr
AI under conditions of low enzyme concentration has been interpreted as slow DNA cleavage by the Sgr
AI dimer (Daniels et al.
; Park, Stiteler et al.
). The current structure supports this interpretation in that DNA has been cleaved by the enzyme in the crystallization experiment and no indication of any other form beyond dimeric is found. It is possible that the activated form of the enzyme was populated at some point prior to crystallization but did not prevent the capture of a large fraction of the Sgr
AI–DNA complexes in this low-activity dimeric form. Only two Mg2+
ions are located in active site B
; however, the postulated (Dunten et al.
) third Mg2+
-binding site at the M2 site is also occupied in active site A
). This Mg2+
binding site was predicted based on analysis of other nucleases, including the closely related Ngo
MIV (Deibert et al.
; magenta, Fig. 6), and is important in the two-metal-ion mechanism model thought to be operative in this and many other divalent cation-dependent nucleases (Horton & Perona, 2002
; Dupureur, 2008
; Etzkorn & Horton, 2004b
; Fig. 7). The high resolution of the structure with cleaved primary-site DNA and Mg2+
(2.20 Å) allows the unambiguous identification of the Mg2+
ions through their short ligation distances to O atoms (relative to those of water) and their octahedral ligation geometry (as opposed to tetrahedral for water) (Fig. 5
). All three sites are occupied in this structure with similar temperature factors and therefore partial occupancy of M2 and M3 is not indicated, although still possible. Three metal-ion sites were also located in Eco
RV, although they were not observed to be simultaneously occupied in any one structure, and a moving metal-ion mechanism has been proposed in which an ion occupying a distal site moves more proximal to the DNA (Horton & Perona, 2004
). Such a mechanism is also possible in Sgr
AI as a mechanism of activation of DNA cleavage by the destabilization of Mg2+
binding to M3 in favor of M2. However, the current structure is thought to be representative of the low-activity conformation and therefore the absence of Mg2+
binding at M2 in one of the two active sites in the asymmetric unit is consistent with this proposal involving weak Mg2+
binding at M2 in the low-activity conformation. Interestingly, a global analysis of DNA cleavage by the type II restriction endonuclease Pvu
II indicated that cleavage with only a single metal ion per active site is ~100-fold slower than that with two metal ions (Xie et al.
II has only two active-site metal-ion binding sites, corresponding to sites M1 and M2 of Sgr
AI (Horton & Cheng, 2000
). We have found that the single-turnover DNA-cleavage rate constant of Sgr
AI for primary-site DNA in the unstimulated form is ~200-fold lower than that in the stimulated form. The similarity in degree of activation of Sgr
AI to that of Pvu
II is further support for the idea that DNA cleavage by Sgr
AI in the unstimulated (i.e.
low-activity) form results from a mechanism that utilizes only a single Mg2+
ion (at M1), while that by the stimulated form results from the utilization of Mg2+
ions at both M1 and M2. The M3 site may be present to decrease Mg2+
binding at M2 and/or as a reserve for Mg2+
when activation (in which the Mg2+
affinity of the M2 site is increased) occurs.
The two-metal-ion mechanism for DNA cleavage by the low-activity conformation of SgrAI. Water molecules that are not directly involved in the reaction are shown as gray spheres.
The structure with cleaved primary-site DNA shows that the cleaved primary site is not sufficient to shift the conformation of Sgr
AI into the activated conformation, since the conformation of Sgr
AI bound to cleaved primary-site DNA is the same as that with uncleaved primary-site DNA (Dunten et al.
) and with secondary-site DNA (current work), assigned as the low-activity conformation. This conclusion is consistent with biochemical data that showed that for stimulation to occur at 310 K the DNA requires sufficient flanking base pairs (Park, Stiteler et al.
) that are absent from the DNA used in these crystal structures. The DNA used in the crystal structures (18 bp) fails to stimulate Sgr
AI in DNA-cleavage assays performed at 310 K, but does stimulate DNA cleavage at 277 K (Park, Stiteler et al.
), where oligomers (HMWS) of the DNA-bound Sgr
AI dimer are also observed (Park, Stiteler et al.
). The HMWS have been proposed to be the activated form of the enzyme (Park, Stiteler et al.
). The temperature of crystallization, 290 K, may be too high to stabilize the oligomeric form of Sgr
AI with this DNA, resulting in the capture of the low-activity dimeric form. The factors that favor the activated form, which is presumably the HMWS, await future structural characterization.