Conservation of structure and function among tyrosine recombinases: homology-based modeling of the lambda integrase core-binding domain

Abstract

Tyrosine recombinases participate in diverse biological processes by catalyzing recombination between specific DNA sites. Although a conserved protein fold has been described for the catalytic (CAT) domains of five recombinases, structural relationships between their core-binding (CB) domains remain unclear. Despite differences in the specificity and affinity of core-type DNA recognition, a conserved binding mechanism is suggested by the shared two-domain motif in crystal structure models of the recombinases Cre, XerD and Flp. We have found additional evidence for conservation of the CB domain fold. Comparison of XerD and Cre crystal structures showed that their CB domains are closely related; the three central alpha-helices of these domains are superposable to within 1.44 A. A structure-based multiple sequence alignment containing 25 diverse CB domain sequences provided evidence for widespread conservation of both structural and functional elements in this fold. Based upon the Cre and XerD crystal structures, we employed homology modeling to construct a three-dimensional structure for the lambda integrase CB domain. The model provides a conceptual framework within which many previously identified, functionally important amino acid residues were investigated. In addition, the model predicts new residues that may participate in core-type DNA binding or dimerization, thereby providing hypotheses for future genetic and biochemical experiments.

Structure-Function Analysis of IntDOT

“…Cre was selected for model building because (i) it provided a low overall threading energy, (ii) known active-site residues were aligned correctly (except for Arg I; see below), (iii) insertions and gaps in the alignment did not disrupt secondary-structure elements that are strongly conserved among tyrosine recombinases (33), (iv) cocrystal structures with Cre allow interactions between the modeled protein and a bound DNA site to be investigated, and (v) we previously obtained good results using Cre to model the IntDOT CB domain (29). Although Cre shares low sequence identity with the IntDOT CAT domain (12.1%), we found previously that modeling of tyrosine recombinases is useful at this level of identity (29,40). However, the model has certain limitations; for example, the true path of the polypeptide backbone may differ by as much as 3 to 4 Å (on average) from the model, and exact conformations of amino acid side chains in variable protein regions (e.g., on DNA-binding surfaces) cannot be predicted reliably (40).…”

“…Although Cre shares low sequence identity with the IntDOT CAT domain (12.1%), we found previously that modeling of tyrosine recombinases is useful at this level of identity (29,40). However, the model has certain limitations; for example, the true path of the polypeptide backbone may differ by as much as 3 to 4 Å (on average) from the model, and exact conformations of amino acid side chains in variable protein regions (e.g., on DNA-binding surfaces) cannot be predicted reliably (40). Nevertheless, the strong conservation of CAT domain structure and active-site configurations among tyrosine recombinases (33,45) suggests that the model can provide a useful approximate representation of the overall IntDOT CAT domain and of the positions of its active-site residues within this structure.…”

“…We previously predicted that the IntDOT CB domain comprises residues 108 through 220 and contains four major ␣-helices (A, B, C, D) (29), arranged in the orthogonally crossed conformation that is typical of tyrosine recombinases (40). It is not clear whether the IntDOT CB domain also contains a fifth ␣-helix (E, near residues 209 to 213) similar to those found in Cre (19) and Flp (7); therefore, in this report we have not labeled any of the helices "E," in order to avoid conflicts in nomenclature.…”

“…A 3-dimensional structure for the CAT domain of IntDOT was produced essentially as described previously (29,40). Briefly, the mGenTHREADER fold recognition algorithm (24, 31) was used to identify sequence-structure alignments between the amino acid sequence of the IntDOT CAT domain and experimentally determined structures in the Protein Data Bank (PDB).…”

Control and Regulation of KplE1 Prophage Site-specific Recombination

“…To check if intS was essential for KplE1 excision we constructed an intS-defective strain (LCB1024). The E. coli K12 genome contains 10 putative integrase genes of the tyrosine recombinase family (30,31), and in our in vivo assay only overexpression of the torI gene was sufficient to promote KplE1 excision (27). It could therefore be argued that either one of the integrase gene products could be involved in the excision reaction, because they belong to the same recombinase family.…”