Flexibility in DNA recombination: structure of the lambda integrase catalytic core - PubMed (original) (raw)

Flexibility in DNA recombination: structure of the lambda integrase catalytic core

H J Kwon et al. Science. 1997.

Abstract

Lambda integrase is archetypic of site-specific recombinases that catalyze intermolecular DNA rearrangements without energetic input. DNA cleavage, strand exchange, and religation steps are linked by a covalent phosphotyrosine intermediate in which Tyr342 is attached to the 3'-phosphate of the DNA cut site. The 1.9 angstrom crystal structure of the integrase catalytic domain reveals a protein fold that is conserved in organisms ranging from archaebacteria to yeast and that suggests a model for interaction with target DNA. The attacking Tyr342 nucleophile is located on a flexible loop about 20 angstroms from a basic groove that contains all the other catalytically essential residues. This bipartite active site can account for several apparently paradoxical features of integrase family recombinases, including the capacity for both cis and trans cleavage of DNA.

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Figures

Fig. 1

Fig. 1

The lambda integrase catalytic domain is a seven-helix bundle cradled by a β sheet and two β hairpins. The ribbon diagram (A) is colored with a gradient from the NH2-terminus (blue) to the COOH-terminus (red) with the program SETOR (58). (B) Topology diagram of the Int catalytic domain with the flexible loop connecting helix G and strand β6 depicted by spheres. The α helices of Int c170 are intertwined in an irregular packing arrangement that, to our knowledge, has not been seen before. (C) The catalytically essential residues Arg212, His30*, and Arg311 face a small depression in the protein surface, located far from the attacking nucleophile Tyr342. Tyr342 is at the junction of the flexible loop (spheres) and strand β6, which forms a hairpin structure with β7 that is, in turn, anchored to the protein core by hydrophobic and electrostatic interactions.

Fig. 2

Fig. 2

(A) Conserved residues cluster around the active site pocket of lambda integrase. These include the Arg212-His308-Arg311 triad (navy), Lys235 and Lys239 (cyan), His333 (magenta), Ser312-Gln328 (green), and Leu331-Gly332 (yellow). Tyr342 (red) is located some distance from the other catalytic residues. (B) Sequence alignment of selected integrase family members showing the conserved hydrophobic residues (brown) that form the core of the Int catalytic domain. This conservation of buried residues strongly implies that integrase family recombinases have similar folds. The other highly conserved motifs (1, 2) are predominantly surface residues that cluster around the enzyme active site, and they are color-coded as for (A). The AMPS program suite (26) was used for alignment of integrase sequences, and (B) was prepared with ALSCRIPT (59). Residue numbers refer to λ Int residues and the secondary structure of the Int catalytic domain is shown. The aligned sequences are: λ, phage lambda Int; HK022, phage HK022 Int; HP1, Haemophilus influenzae phage HP1 Int; P2, phage P2 Int; XerD, E. coli XerD; L5, Mycobacterium spp. phage L5 Int; Tn1545, Streptococcus pneumoniae transposase Tn1545; P22, phage P22 Int; P1, Cre of phage P1; SSV1, Sulfolobus phage SSV1; mcoc, Methanococcus jannaschii putative Int; Flp, Saccharomyces cerevisiae Flp.

Fig. 3

Fig. 3

Theoretical model of the λ Int catalytic core bound to a B-form half-att site. A full att site contains a pair of inverted core-type Int binding sites. An Int protomer at each site is responsible for cleaving one DNA strand via formation of a covalent 3′ phospho-tyrosine linkage and a free 5′-hydroxyl. The two nicks are staggered by seven base pairs with a 5′ overhang. For clarity, only one subunit of the Int c170 dimer that was modeled on DNA is shown. The catalytic Arg-His-Arg triad (cyan) of Int is docked over one of the scissile phosphates (shown as breaks in the DNA ribbon). The Cα trace of Int c170 (blue) is displayed with the active site loop containing the Tyr342 nucleophile shown in two alternative conformations. The orientation corresponding to cis cleavage (orange tyrosine) is a theoretical model, whereas that corresponding to trans cleavage (red tyrosine) is present in one of two Int protomers in the crystal structure. The segment of the loop that is disordered in both protomers (Lys334 to Gln341) is modeled in pink.

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