Structural Basis for Catalytic Activation of a Serine Recombinase

Abstract

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here we present a 1.86Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

Arginine as a General Acid Catalyst in Serine Recombinase-mediated DNA Cleavage

“…These key residues cluster near the catalytic serine in several crystal structures of serine recombinases (15,19,20). The structure that appears closest to a catalytically competent state is that of the tetrameric Sin Q115R catalytic domain, in which all of the key residues form a hydrogen-bonded network surrounding a sulfate ion that lies in the scissile phosphate-binding pocket (16) (Fig. 1, B and C).…”

“…Although WT resolvases usually require accessory factors to trigger tetramerization, the pseudo-WT Sin used here (Q115R/R54E) does not. B, structure of the tetrameric catalytic domain of Sin Q115R/R54E (Protein Data Bank ID code 3PKZ) (16). The nucleophilic serine is shown as red spheres, and bound sulfate ions as sticks.…”

“…C, active site of Sin. Stereo view shows the Sin active site from the structure in B, in which all of the conserved residues implicated in catalysis form a hydrogen-bonded network surrounding a sulfate ion that lies in the scissile phosphate group-binding pocket (16). The path of a modeled DNA backbone is sketched as a yellow line.…”

A possible way that φC31 integrase regulates the recombination direction

“…isolated several mutated integrases that gain the ability to recombine attL and attR without affecting the attB×attP recombination. Interestingly, these mutants are all located in a putative coiled-coil region of CTD , rather than NTD like in serine resolvases (Burke et al, 2004;Li et al, 2005;Keenholtz et al, 2011). The putative coiled-coil structure contains two regions, E449-D477 and R489-A519 , which are termed C1 and C2, respectively, in this article.…”

“…Aside from φC31 integrase, some serine resolvases can also be activated by mutations (Burke et al, 2004;Li et al, 2005;Keenholtz et al, 2011). Their crystal structures have been well determined (Li et al, 2005;Keenholtz et al, 2011), pointing to the same activating principle that most of the mutations tend to stabilize the resolvase tetramer or destabilize its dimeric form (Li et al, 2005;Keenholtz et al, 2011).…”