Ignment of fungal HET domains with TIR domain proteins.The TIR domains of two bacterial proteins of recognized structure and from the human TLR TIR domain PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21501643 (boxed in red) are aligned with the HET domains of P.anserina HETe and Neurospora crassa TOL (boxed in blue) with each other with related sequence of diverse phylogenetic origin annotated as HET domains in Pfam.On top in the alignment, the components of secondary structure of Brucella TcpB are shown.Sequence designations are as follows Paracoccus, Paracoccus denitrificans, gij; Brucella, Brucella A-196 Epigenetics melitensis, gij; Human Tlr, Homo sapiens, gij; Candidatus, Candidatus Accumulibacter, gij; Emiliania, Emiliania huxleyi, gij; Ectocarpus, Ectocarpus siliculosus, gij; Thalassiosira, Thalassiosira pseudonana, gij; Salpingoeca, Salpingoeca rosetta, gij, Physcomitrella, Physcomitrella patens, gij; Podospora, P.anserina, gij (HETe); Neurospora, Neurospora crassa, gij (TOL).Table Repeat number polymorphism in ANK and TPR Repeat Domains of NLR Proteins from Podospora anserinaPa__ PNPUDP NACHT ANK S Wa Wa Wa Wa Wa Total Distinctive ND Pa__ PNPUDP NACHT ANK Pa__ sesBlike NBARC TPR ND Pa__ PFD NBARC TPR ND Pa__ PFD NBARC TPR ND Pa__ sesBLike NACHT TPR(HEAT) ND Pa__ sesBlike NACHT TPR ND Pa__ UNK NBARC TPR ND NOTE.ND, not determined.3 forms of superstructureforming repeats, WD, ANK, and TPR motifs.We have previously shown that WD repeats of NLRlike proteins show substantial repeat size polymorphism in Podospora and are topic to concerted evolution and positive diversifying choice (Paoletti et al.; Chevanne et al).We extended this analysis to ANK and TPR motif NLR proteins of Podospora, so that you can identify regardless of whether repeat size polymorphism and diversifying selection was a typical property of such repeat domains.We selected eight P.anserina NLRencoding genes showing hugely conserved ANK and TPR motifs, and PCRamplified the repeat region from genomic DNA from 5 unique wild isolates.For every single locus, sequence evaluation revealed repeat number polymorphism (RNP) (table).ANK repeat numbers ranged from to above , whereas TPR motif numbers ranged from to above .The RNPs observed suggest frequent recombination amongst repeats inside a locus, and possibly involving loci encoding the same type of repeats, as previously reported for WDrepeats (Paoletti et al.; Chevanne et al).Subsequent, we chosen 1 ANK repeat locus and a single TPR motif locus for which we had sequenced the highest number of repeats (Pa__ and Pa__, respectively) andanalysed the variability of your repeats from person loci.For every locus, individual repeat sequences had been aligned and analysed for position below optimistic selection (see Materials and Techniques) (fig).5 positions showed signs of positive selection within the ANK repeats and 3 within the TPR motifs.To find the positive selection and polymorphic web-sites on the repeat domain structure, the repeats had been homologymodeled to ANK and TPR domains of identified structure.The TPR motif domain of Pa__ was modeled making use of the human kinesin light chain structure (Protein Data Bank [PDB] ID EDT) as template.Inside the TPR motifs, all constructive selection internet sites also as the other polymorphic position mapped towards the concave side of your TPR structure inside the ahelical regions.The ANK repeat domain of Pa__ was modeled employing the structure of artificial ANK repeat domain of engineered protein OR (PDB ID GPM) as template.In the ANK repeats, with 1 exception, the positive selection and polymorphic web-site also mappe.
