1. Volume 12, Issue 1, Pages 187-198 (July 2003)
A Conformational Switch Controls the DNA Cleavage Activity of λ Integrase 
Hideki Aihara, Hyock Joo Kwon, Simone E. Nunes-Düby, Arthur Landy, Tom Ellenberger 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Crystallization of λ Int Complexed to a DNA Recombination Intermediate
(A) A suicide DNA substrate containing a trinucleotide flap was used for crystallization with λ Int. The flap prevented hydrolysis of the covalent phosphotyrosine intermediate between Y342 of λ Int and the 3′ end of the cleaved DNA (see text for details).
(B) The experimental electron density for the λ Int C75-DNA complex was readily interpretable after solvent flattening. The axis of the DNA double helix (red) is oriented horizontally, with the major and minor grooves readily evident. This view is oriented similarly to the ribbon diagram shown in Figure 2A (right panel). The figures were produced with the following molecular graphics programs: PYMOL ( for Figure 1B, RIBBONS (Carson, 1991) for Figure 2A, MOLMOL (Koradi et al., 1996) for Figures 2B and 5B, MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997) for Figures 2C, 3, 4, and 5A.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 λ Int's Central and Catalytic Domains Encircle DNA
(A) Ribbon diagrams showing the central domain (residues 75–160; above the DNA) and the catalytic domain (residues 170–356; below the DNA) of λ Int, and their interactions with the major and minor grooves on the opposite sides of the DNA. A long, extended linker (residues I160–R176) connects these domains. The scissile phosphate that is covalently linked to Y342 is shown as a red sphere. The central domain inserts into the major groove adjacent to the site of DNA cleavage. The catalytic domain makes interactions with the major and minor groove on the opposite side of the DNA, straddling the site of DNA cleavage.
(B) The solvent accessible surface of the Int protein is shown, colored according to electrostatic potential. The DNA binding surface is highly positive (blue) and makes numerous interactions with the phosphates of the DNA (cf. Figure 3B). The polypeptide linker between domains joins the central and catalytic domains on one side of the DNA. A salt bridge between the Nζ of K93 and the carbonyl oxygen of S234 bridges between domains on the other side of the DNA, completing the ring-shaped structure that encircles the DNA.
(C) The architecture of the λ Int C-75 protein is shown with cylinders and arrows representing helices and β strands, respectively. This view is oriented similarly to that in (A) (right side). The central domain of λ Int lacks helix E, corresponding to the fifth helix of Cre's N-terminal domain, which is involved in subunit interactions. The secondary structures of the λ Int C-75 protein are assigned as follows: αA(76–87), αB(94–110), αC(121–133), αD(137–156), αF(182–194), αG(198–209), αH(213–218), β1(224–225), β2(228–232), β3(239–243), β4(247–248), β5(253–254), αI(255–265), αJ(282–296), αK(309–321), αL(324–331), β7(349–353).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 DNA Contacts between λ Int and the Core-Type att Binding Site
(A) The side chains of the central domain (top of figure) and of the catalytic domain (bottom of figure) make relatively few base-specific interactions in the major groove of the att site DNA, consistent with the weak binding specificity of λ Int (Ross and Landy, 1983). Residues N99 and K95 from helix αD of the central domain make several hydrogen bonding contacts to the DNA near the site of cleavage. Helix αJ of the catalytic domain inserts into the major groove at the center of a bend in the DNA (cf. Figure 2A).
(B) A schematic illustration of contacts between λ Int and the core-type att sequence. Hydrogen bonding and other electrostatic interactions are depicted by black lines (solid lines, distances < 3.3 Å; dashed lines, 3.3 Å ≤ distances < 3.6 Å), and van der Waals contacts to the C5 methyl groups of thymines are shown by cyan dashed lines. Residues from both the central (orange) and catalytic (purple) domains make many contacts to the phosphate backbone, whereas only a few direct hydrogen-bonding interactions to the DNA bases are seen (bases colored green). Contacts mediated by main chain atoms are indicated by the prefix “mc.”
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
The Active Conformation of λ Int Is Stabilized by Interactions with DNA
(A) The invariant catalytic residues R212, H308, and R311 coordinate the scissile phosphate at the site of DNA cleavage. The Y342 nucleophile for DNA cleavage is covalently bonded to the 3′-phosphate of the cleaved DNA. K235 is a residue that is widely conserved in tyrosine recombinases and type IB topoisomerases (Cheng et al., 1998) (cf. Figure 3A).
(B) In the active conformation of λ Int, Y342 has moved more than 20 Å in comparison to its position in the unbound structure of Int (Kwon et al., 1997). The Y342 nucleophile is held in this position by the covalent linkage with the cleaved 3′ end of the DNA and by numerous electrostatic interactions shown here. These include interactions with the bound DNA and contacts by residues of the Y342 loop to the invariant core of the catalytic domain. The repacking of the Y342 loop in structures of free and DNA-bound λ Int constitutes a conformational switch that modulates DNA cleavage activity.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
A Remodeling of Int's Active Site Switches DNA Cleavage Activity On and Off
(A) A comparison of the DNA-bound (left) and unbound (right) structures of λ Int shows a dramatic reorganization of the C-terminal region spanning residues 331–356 (red). In the absence of DNA, Y342 (yellow) is far from the catalytic triad of R212, H308, and R311 (magenta side chains). In the DNA complex (left panel), Y342 has moved into the active site. Another consequence of the DNA-bound conformation is that the extreme C-terminal residues 349–356 extend away from the parent Int molecule and pack against another molecule in trans as shown in (B) and (C).
(B) The C-terminal tail of λ Int spanning residues 349–356 is a protein-protein interaction motif. In the active form of the Int recombinase, the C-terminal residues mediate a protein interaction in trans with a neighboring molecule (red circle). The right panel shows the trans-packing interactions of the C-terminal tail of one molecule (yellow bonds) against the surface of another molecule (colored by electrostatic potential). The side chain of I353 plugs into a pocket of the underlying surface. The mutation of I353 to methionine results in increased DNA cleavage activity and a loss of normal recombination functions (Kazmierczak et al., 2002; Tekle et al., 2002).
(C) The interactions of the C-terminal tail are nearly identical in the trans-packing arrangement between adjacent molecules of λ Int complexed to DNA (left panel) and the inactive, cis-packing interactions seen in the unliganded conformation of the recombinase (right panel). In the absence of DNA, the cis-packing arrangement of the tail anchors Y342 away from the active site. Numerous contacts between residues 349–356 and the underlying residues of the catalytic domain secure the tail in both the cis and trans conformations. In both conformations, the side chain of W350 packs between the side chains of Y230 and K239. Strand β7 adds on to the β sheet formed by β1-β3. The functionally important side chain of I353 (Tekle et al., 2002) makes numerous van der Waal contacts with the underlying surface of the catalytic domain, securing the C-terminal tail.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 A Conformational Switch in λ Int
This cartoon illustrates the DNA-bound conformation of λ Int that positions Y342 for cleavage of DNA. This isomerization from the inactive form, in which Y342 is held some distance from the catalytically important Arg212-His308-Arg311 triad (Kwon et al., 1997), to the active conformation seen in complex with DNA is accompanied by the release of strand β7 and its repacking in trans against a neighboring molecule. In higher order recombination complexes, trans interactions of the C-terminal among neighboring DNA-bound subunits could modulate the activities of individual subunits.
Molecular Cell  , DOI: ( /S (03) )