Folding, DNA recognition, and function of GIY-YIG endonucleases: crystal structures of R.Eco29kI

Abstract

The GIY-YIG endonuclease family comprises hundreds of diverse proteins and a multitude of functions; none have been visualized bound to DNA. The structure of the GIY-YIG restriction endonuclease R.Eco29kI has been solved both alone and bound to its target site. The protein displays a domain-swapped homodimeric structure with several extended surface loops encircling the DNA. Only three side chains from each protein subunit contact DNA bases, two directly and one via a bridging solvent molecule. Both tyrosine residues within the GIY-YIG motif are positioned in the catalytic center near a putative nucleophilic water; the remainder of the active site resembles the HNH endonuclease family. The structure illustrates how the GIY-YIG scaffold has been adapted for the highly specific recognition of a DNA restriction site, in contrast to nonspecific DNA cleavage by GIY-YIG domains in homing endonucleases or structure-specific cleavage by DNA repair enzymes such as UvrC.

Figure 1. The structure of the unbound R.Eco29kI restriction endonuclease

Panel a: The enzyme homodimer. Subunit 1 is colored by secondary structure; subunit 2 is colored light blue. The GIY-YIG domain of each subunit is a β-sandwich fold containing a 3-stranded antiparallel β-sheet and two α-helices. In R.Eco29kI this core is interrupted by a short additional helix (α2) located between the first and second β-strand (residues 48 to 148; core topology β1–α 2–β 2–α 3–β 3–α4). This region is also interrupted by a poorly ordered surface loop (L1, residues 81 to 98) that packs against the center of the DNA recognition site in its minor groove. The C-terminal region (residues 149 to 214) contributes two additional exposed loops (L2 and L3) to the DNA-binding interface, and a final helix (α5) at the protein carboxy-terminus. Panel b: Topology diagram of an R.Eco29kI subunit. The secondary structure is colored and labeled as in panel a. The L1, L2 and L3 loops are colored blue and labeled. The residues of the GIY-YIG motif and additional active site residues are indicated in red font. Three residues involved in base-specific DNA recognition are indicated in blue font. The domain-swapped N-terminal extention (residues 1 to 14) is indicated in green. Panel c: The protein dimer viewed from the top, to illustrate the structural basis of protein dimerization. The domain-swapped N-terminal extensions of each subunit (residues 1 to 14) are blue and green. The α1 helix continues from that extension, and is bundled against it α1’ symmetry mate. These two helices lie across one face of the α4-α4’ helical bundle to form the majority of the homodimeric protein-protein interface. See also Supplementary Figure S1.

Figure 2. Structure of the DNA-bound R.Eco29kI endonuclease

Panel a: The bound enzyme. The two subunits rotate by ~ 20° towards one another to engage the DNA duplex; the six basepair restriction site is completely encircled by the exposed loops of the enzyme homodimer. The dimensions of the enzyme are reduced by ~ 10 Å in the longest dimension. The DNA duplex is almost completely unperturbed from canonical B-form duplex conformation as a result of binding. Panel b: Superposition of a bound and unbound R.Eco29kI subunit (superposition calculated using the GIY-YIG core domain described above); the rmsd for backbone atoms across this domain is ~1.7 Å. The domain closure shown in panel b is accommodated by a hinge movement in the N-terminal region of the protein at residues 14/15 (arrow). In addition, the L1 loop in the GIY-YIG domain reorganizes to establish contacts to the minor groove at the center of the restriction site (see Figure 3). See also Supplementary Figure S3.

Figure 3. Diversity of GIY-YIG endonucleases

Panel a: Structure-based sequence alignment of five GIY-YIG endonucleases that have been visualized using x-ray crystallography (R.Eco29kI, UvrC, I-TevI and T4 Endonuclease II) or solution NMR (Slx-1). The sequences of two additional GIY-YIG restriction endonucleases that are both isoschizomers of R.Eco29kI, but that act as tetramers instead of homodimers, are also shown (R.NgoMIII and R.Cfr42I). The secondary structure of R.Eco29kI is shown above the alignment. Black residues correspond to the core GIY-YIG fold (spanning β1, β2, α3, β3 and α4). Additional secondary structural elements that are unique to one or more members of the alignment are green; the DNA-binding loops of R.Eco29kI are blue, and the six residues of the GIY-YIG motif are red and boxed. Residues found in the R.Eco29kI active site are denoted with bold font and asterisks; all are conserved across all the enzymes shown except for N154 (which is missing in Slx-1). Residues that make sequence-specific contacts to DNA bases are indicated with bold font and dots; all are conserved across the three restriction endonucleases. Panel b: Structural alignment of the endonuclease domains shown in panel a above. All alignments were performed using the β1, β2 and β3 strands at the core of the GIY-YIG domain as the structural basis for superpositions. The overall alignment of the core domain of all five endonucleases is shown in the box; all of the core folds with the exception of Slx-1 superimpose with rmsd for backbone atoms of approximately 2.7 Å. The latter fold (colored light cyan for uniqueness) is considerably more divergent (rmsd ~ 3.6 Å). Within the core fold, there is one unique inserted secondary structural element (α2 in R.Eco29kI, which is inserted between β1 and β2). As well, the α3 helix of Slx-1 occupies a significantly different position relative to the equivalent region in the related proteins. The additional ribbon diagrams display the individual complete subunits or domains of each endonuclease from the superposition. The coloring of secondary structural elements is identical to that used in panel a above. See also Supplementary Figure S6.

Figure 4. DNA recognition and binding by R.Eco29kI

Panel a: The endonuclease homodimer completely encircles the DNA target, as also illustrated in Figure 1b. Residues from loops L1 and L2 in each subunit (R86, H163 and R169), as well as backbone atoms from neighboring residues 164 and 165 in L2, form sequence-specific contacts to DNA bases. The contacts to bases in each DNA half-site correspond to residues from individual protein subunits (right side of panel a). Panel b: Illustration of DNA recognition viewed looking down the axis of the DNA duplex. Left: The endonuclease homodimer in the bound conformation (viewed without the DNA), with residues from the L1 loop (R86 and R86’) and the L2 loop (H163 and H163’; R169 and R169’) shown and labeled. R86 and its L1 loop interact with each DNA half-site the minor groove; H163 and R169 and the L2 loop interact with the major groove. Right: Contacts made to basepairs -1, -2 and -3, shown in the same orientation as the protein in the left side of the same panel. The numbering of individual bases, as well as the center of the palindromic site and the positions of cleavage on each strand, is shown to the left. Readout of the individual bases involves (1) direct contacts between R169 and Gua3 in the major groove for basepairs +/−1; (2) direct contacts between backbone oxygens of residues 164 and 165 and Cyt 2 in the major groove for basepairs +/−2 (augmented by a water-mediated contact between the same base and R86 in the minor groove); and (3) a direct contact between H163 and Gua6 in the major groove for basepairs +/−3. See also Supplementary figure S4.

Figure 5. The R.Eco29kI active site

Cleavage occurs between Cyt4 and Gua5 on each strand of the restriction site. Panel a: The scissile phosphates are located 15 Å apart, entirely within separate active sites. All residues in each active site are provided entirely by individual protein subunits. Panel b: The active site of one protein subunit, shown in two different orientations (rotated by ~90° relative to one another). In the left image the view is looking across the DNA strand; in the right image the view is looking down the same DNA strand (from the approximate position of the incoming water nucleophile). The residues shown and labeled correspond to those conserved residues also found in the active sites of UvrC, I-TevI, and T4 endonuclease II (Slx-1 is missing N154). E142Q denotes the inactivating point mutation included in the construct of R.Eco29kI used for these structural studies. A single bound water molecule is clearly visible in-line with the scissile phosphate, giving a ‘through phosphorus’ angle to the 3′ oxygen leaving group of ~ 180°; the distance from this water to the phosphorus and to the phenolate oxygen of Y49 is approximately 3 Å. H108 is not located close enough to act directly on the water molecule, but is positioned appropriately to assist in acid base catalysis by establishing an H-bond to Y49. E142 and N154 are found at positions that could bind a divalent metal ion in coordination with a nonbridging oxygen of the scissile phosphate. Panel c: Comparison of the GIY-YIG endonuclease active site of R.Eco29kI (left) with the HNH active site of R.Hpy99I (right), viewed from the same relative orientation of the scissile phosphate and flanking bases. The proposed mechanism and overall catalytic architecture is largely the same (with the exception of the substitution of a tyrosine for a histidine), but are grafted onto two completely separate protein fold topologies. The sphere in the R.Hpy99I structure is a bound magnesium ion; the open circle in the R.Eco29kI active site represents the equivalent position, which would allow coordination by N154, E142Q and the scissile phosphate. See also Supplementary Figure S5.

Figure 6. The GIY-YIG active site and proposed mechanism

Panel a: Structure superposition of the crystallized GIY-YIG endonucleases, color coded as shown in the table of homologous residues. The scissile phosphate and flanking bases from R.Eco29kI are shown for orientation purposes. The active site from both subunits of the R.Eco29kI are shown (dark and light blue). The positions and identity of four residues (corresponding to Y49, Y76, E142 and N154) are well conserved (except for the absence of the Asn residue in Slx1). In contrast, the conformation and/or identity of residues corresponding to H108 and R104 are more divergent. Panel b: Proposed mechanism of R.Eco29kI. The structure indicates that strand cleavage in each active site involves a single bound metal ion (coordinated by at least E142, and possibly by N154) and the activation of a water molecule by a tyrosine residue. In this figure, we indicate Y49 as the likely general base. However, the slightly different positions and relative angles of the active site tyrosines, waters and phosphate in the separately visualized active sites in R.Eco29kI do not currently allow us to unambiguously assign this role to Y49, Y76, or a combination of the two residues.