1. Volume 14, Issue 5, Pages 869-880 (May 2006)
The Structure of I-CeuI Homing Endonuclease: Evolving Asymmetric DNA Recognition from a Symmetric Protein Scaffold 
P. Clint Spiegel, Brett Chevalier, Django Sussman, Monique Turmel, Claude Lemieux, Barry L. Stoddard 
Structure 
Volume 14, Issue 5, Pages (May 2006)
DOI: /j.str
Copyright © 2006 Elsevier Ltd Terms and Conditions

2. Figure 1
Phylogenetic Location and Structures of Algal Homing Endonucleases in This Study
(A) Distribution of group I introns and homing endonuclease ORFs in the chloroplast LSU rRNA gene from various green algae. Hosts containing HEs of the I-CeuI family are restricted to the advanced class Chlorophyceae, whereas hosts containing HEs of the I-CreI/I-MsoI family are found in all four classes of the Chlorophyta. Only the nodes supported by more than 50% bootstrap values are shown; the nodes with less than 50% support have been collapsed. The subfamilies of the group I introns identified in the chloroplast large subunit rRNA gene of green algal taxa are denoted by symbols (black triangles: IA1 introns; black squares: IA3 introns; black circles: IB4 introns). The number associated with each insertion site corresponds to the position of the introns relative to E. coli 23S rRNA. Filled symbols represent introns hosting a putative HE gene. HEs whose activities have been experimentally demonstrated are denoted by asterisks. The I-CeuI, I-CreI, and MsoI HEs are denoted by the “cross” symbol and by red boxes.
(B) DNA bound complex structures of I-CeuI (left) and I-CreI/I-MsoI (right).
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

3. Figure 2
Sequence Alignment of I-CreI, I-MsoI, and I-CeuI Proteins and DNA Targets Sites
(A) The overall pairwise sequence identity between the enzymes is shown to the left of the alignment; the secondary structure elements for I-CeuI are shown above the alignment. Residues conserved between all three enzymes are red; those conserved between any two enzymes are blue. The LAGLIDADG motif is outlined; the metal binding acidic residue is indicated with an arrow. The α helices are numbered according to convention, and the helix containing the endonuclease core motif is denoted as “α1.” The N-terminal helices that form a unique structural elaboration in I-CeuI are denoted as “α-2” and “α-1.” Residues in I-CeuI involved in DNA contacts to the major groove base edges and to the phosphate backbone are denoted by black circles; residues involved in additional contacts to base edges in the minor groove are denoted by green circles. The alignment was generated by using CLUSTALW (Thompson et al., 1994).
(B) The bases that are conserved between the left and right half-sites of each target are underlined. Bases conserved across all three sites are red; those conserved between any two sites are blue. The cleavage pattern of each enzyme is shown by staggered lines. The overall identity between sites is shown.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

4. Figure 3 Superpositions of I-CeuI and I-CreI
(A and B) Superposition of I-CeuI is shown in green, and superposition of I-CreI is shown in blue. Superpositions shown from the (A) side and (B) bottom of the enzyme. The LAGLIDADG helices are shown in the same orientations to the right. The rmsd for backbone atoms of individual subunits is ∼2 Å. The relative orientation of the two DNA-contacting β platforms, calculated from the bottom of the conserved LAGLIDADG helices, differs by ∼5° (indicated by a black arrow). This difference is caused by a shift in the packing of the LAGLIDADG helix against the corresponding enzyme core in each subunit (indicated by a red arrow for one subunit), rather than by a rigid body rotation of the two subunits.
(C) Left: Magnification of the superimposed dimer interfaces of I-CeuI and I-CreI, which contain the conserved residues of their respective active sites. Right: The same orientation with only the I-CeuI interface and active sites shown. Catalytic residues of I-CreI are blue, and those of I-CeuI are colored by element type. A single bound calcium ion in the I-CeuI structure is shown; the corresponding anomalous difference density is shown in the right panel. The calcium is bound between the scissile phosphates and the corresponding metal binding residues. The Q93 residue of I-CeuI is modeled from the crystal structure of the Q93R mutant used to solve its structure.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

5. Figure 4
Structural Divergence and Elaboration across the LAGLIDADG Enzyme Family
The endonucleases are arranged from the largest enzyme on top (I-CeuI, a homodimer with two 218 residue subunits; or 436 residues total) to the smallest enzyme on the bottom (I-DmoI, a free-standing monomeric enzyme containing 188 residues). I-CeuI displays two significant elaborations on the core LAGLIDADG fold that increase the total contact area to the DNA target: an N-terminal bundle of two α helices that provide nonspecific polar contacts to the DNA backbone (red arrows; also see Figure 2A), and an extended loop (denoted by blue arrows) between α helices 5 and 6 that provides contacts within the minor groove to bases +/−3 and 4 in each half-site.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

6. Figure 5 DNA Contacts by I-CeuI
(A) The numbering of bases, extending from the center of the four base cleavage site, and the corresponding numbering of the bases in the deposited pdb file. Unambiguous noncovalent contacts to individual bases are shown below. The scissile phosphate groups are red. Base pairs that are conserved between the left and right half-sites, and enzyme residues that are engaged in identical contacts to bases in each half-site, are shaded. Structured water molecules involved in contacts between the DNA target and the enzyme are indicated with circles; the single observed bound metal ion (calcium) is indicated by a circled “M.” This single metal ion is indicated twice in the figure, and it is shared between the scissile phosphates and the enzyme active sites. Residue 93, which is a conserved glutamine in the wild-type enzyme, is present in the structure as a catalytically inactivating arginine (Q93R); this side chain is in contact with the phosphate in each half-site directly 5′ to the scissile phosphate. In structures of I-CreI and I-MsoI, the wild-type glutamine residue participates in coordination of a metal bound water molecule.
(B) Ribbon diagram of the β sheet DNA binding platform and additional elaborations (α-2 and loop 5/6) from I-CeuI; residues participating in DNA-contacts are shown and labeled. The same view of the DNA binding elements of the enzyme with the bound target site is shown below.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

7. Figure 6
Conservation of Residues in the I-CeuI Lineage of Homodimeric Endonucleases and Correlation of DNA-Contacting and Active Site Residues with Mutational Inactivation Data
This figure is modified from Figure 4 of Turmel et al. (1997). The amino acid sequences of nine homing endonucleases that are all encoded within group 1 introns at site 1923 in algal LSU rDNA genes are aligned; residues conserved in this lineage are denoted with white-on-black characters. The sequence of I-CreI (from the lineage found in intron insertion site 2593) is shown below for comparison. Positions of residues involved in DNA recognition contacts are indicated with black spheres; positions of catalytically critical residues in the active site are indicated with arrows and labels (the active site residues of I-CreI are shown with gray arrows and labels below the alignment). Positions of residues that were found to be correlated with reduction or loss of endonuclease function, when substituted in a randomized point mutagenesis screen, are indicated with orange circles.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

8. Figure 7 Asymmetric DNA Binding and Cleavage by I-CeuI
(A) Cleavage of the wild-type I-CeuI target (site 1923 from C. eugametos LSU rDNA) and of two palindromic variants corresponding to inverted repeats of the left DNA half-site (base pairs −1 to −11) and the right DNA half-site (base pairs +1 to +11), respectively. The progression of the reaction, and the DNA bend parameters for the bound wild-type site, are shown to the right.
(B) Superposition of the individual I-CeuI subunits and the β sheet DNA binding platforms from their bound complex with the corresponding individual DNA target half-sites. The protein and/or DNA from the “left” half-site (bases −1 to −11 in Figure 5A) are green; the corresponding structure of the “right” half-site is blue. The rmsd of all backbone atoms is less than 1.0 Å; the rmsd of side chain atoms in the DNA interface is ∼1.7 Å. Accommodation of differences in DNA base pair recognition by the two subunits, leading to binding and cleavage of the asymmetric target site, is almost entirely facilitated by small variations in side chain rotameric conformations and the position and use of bound intermediate solvent molecules (the latter feature is schematized in Figure 4A).
(C) Superposition of the DNA binding platform of the two I-CeuI subunits, shown looking into the face of its DNA-contact surface (left); the same orientation with bound DNA half-sites is shown on the right. The rmsd of bound DNA atoms in the superimposed DNA bound complexes of the half-sites is similar to that of the contact residue side chains, at ∼1.8 Å.
(D) Close up of contacts made between enzyme side chains to base pairs +/−8 and 9 (left) and base pairs +/−5 and 6 (right).
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions