A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch

Characterization of the Monoterpene Synthase Gene tps26, the Ortholog of a Gene Induced by Insect Herbivory in Maize1[W][OA]

Plants damaged by insects can synthesize and release volatile chemicals that attract natural enemies of the herbivore. The maize (Zea mays subsp. mays) terpene synthase gene stc1 is part of that indirect defense response, being induced in seedling blades in response to herbivory by beet army worm. Many genes in maize are duplicated because of a past whole-genome duplication event, and several of these orthologs display different expression patterns. We report here the isolation and characterization of tps26 and confirm by homology and synteny criteria that it is the ortholog of stc1. Prior genetic analysis revealed that the stc1 function is not duplicated, raising the interesting question of how the two orthologs have become differentiated in their expression. tps26 encodes a 633-amino acid protein that is highly conserved with STC1. Like stc1, tps26 is induced by wounding, but in the roots and leaf sheath, instead of the blade, and not in response to beet army worm feeding. tps26 maps near a quantitative trait locus for Southwestern corn borer resistance, making it a plausible candidate gene for that quantitative trait locus. However, while possessing highly polymorphic tps26 alleles, the resistant and susceptible parents of the mapping population do not differ in levels of tps26 expression. Moreover, tps26 is not induced specifically by Southwestern corn borer feeding. Therefore, although they share a wounding response, the stc1 and tps26 maize orthologs differ in their tissue specificity and their induction by insect herbivores. The N termini of STC1 and TPS26 are predicted to encode plastid transit peptides; fusion proteins of green fluorescent protein to either N terminus localized to the plastid, confirming that prediction. The mature proteins, but not the respective complete proteins, were active and synthesized a blend of monoterpenes, indicating that they are monoterpene synthases. A gene closely related to stc1/tps26 is found in the sorghum (Sorghum spp.) genome at a location that is not orthologous with stc1. The possible origin of stc1-like genes is discussed

Schematic diagram of precursor and mature intein modified protein.

<p>The precursor intein modified protein consists of N- and C-extein domains (blue) broken up by an intein insertion domain (red). Active inteins predominately contain a conserved N-terminal cysteine or serine residue (C/S) and a conserved C-terminal asparagine (N). The extein site where the intein is inserted is typically N-terminal to a cysteine, serine, or threonine residue (C/S/T). The extein residues surrounding the intein insertion site (−3 to +4) are designated as the intein insertion site cassette. Upon splicing the protein forms the mature form and the −1 and C/S/T residues at the +1 are covalently joined.</p

Western blot demonstrating intein splicing at the T134 and S158 sites of XynB.

<p>Intein mutagenized XynB:Tth clones were expressed from lamba phage. Phage infected E.coli were grown on xylanase diagnostic plates as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037355#pone-0037355-g006" target="_blank">Fig.6</a>, but at a lower plaque density that allowed scoring individual plaques for xylanase activity. Plaques that displayed distinctively strong blue color, indicative of high xylanase activity, were phagemid rescued to E.coli SOLR cells, and bacterium lysates expressing intein mutagenized XynB:Tth was Western blotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037355#pone-0037355-g005" target="_blank">Figure 5</a>. Duplicate samples (A and B) of two XynB:Tth clones with mutagenized intein inserted at the T134 and S158 sites are shown at the left. Empty vector control (−) and the intein unmodified XynB is at the right. The T134 and S158 site both support splicing.</p

Analysis of XynB C/S/T sites for features seen in native insertion sites.

†<p>Distance to secondary structure-loop junction is the number of sites away in primary sequence.</p

Structure based native-like features of intein insertion sites.

<p>(<b>a</b>) Local secondary structure of intein insertion sites binned into 6 different groups relative to its location to loop/alpha-helix and loop/beta-sheet junctions. These groups are AH/L-J (within 2 amino acids of an alpha-helix/Loop junction), BS/L-J (within 2 amino acids of an beta-sheet/Loop Junction), mid-AH (middle of an alpha helix and more than two amino acids to a loop junction), mid-BS (middle a beta-sheet and more than two amino acids to a loop junction) mid-L (middle of a loop and more than 2 amino acids to a loop or beta-sheet junction, and other (all remaining sites). (<b>b–c</b>) Proximity of intein insertion sites to the closest (b) active site residue or (c) dimer interface residue. (<b>d</b>) Degree of burial of intein insertion sites divided by C/S/T residue type. The degree of burial was calculated using the C_β density. (<b>e</b>) Flexibility of the insertion site residue as determined by RMSD of molecular dynamics simulation. This plot shows the flexibility rank of the native versus non-native insertion sites.</p

ROC curve of predictive features for intein insertion sites.

<p>The true positive rate and the false positive rate was calculated for the 4 features: (blue) distance to the active site or dimer interface, (green) SVM score, (red) distance to SS/Loop junction and (cyan) sequence conservation. These rates were determined over a range of cutoff values which were: for the distance to the active site or dimer interface site the cutoff value was varied from 0 to 30 Å, for the SVM scoring the cutoff was varied from −10 to 10, for the distance to the SS/Loop junction the cutoff was varied from 0 to 5 amino acids and for the conservation rank the cutoff was varied from 0 to 1. The points marked with circles are the maximum enrichment points of the true positive rate versus the false positive rate. The black dashed line indicates a random prediction.</p