Ignment of fungal HET domains with TIR domain proteins.The TIR domains of two bacterial proteins of identified structure and on the human TLR TIR domain PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21501643 (boxed in red) are aligned together with the HET domains of P.anserina HETe and D3-βArr Biological Activity Neurospora crassa TOL (boxed in blue) with each other with related sequence of diverse phylogenetic origin annotated as HET domains in Pfam.On best of the alignment, the elements of secondary structure of Brucella TcpB are shown.Sequence designations are as follows Paracoccus, Paracoccus denitrificans, gij; Brucella, Brucella 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 Exclusive 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’ve previously shown that WD repeats of NLRlike proteins show extensive repeat size polymorphism in Podospora and are subject to concerted evolution and positive diversifying choice (Paoletti et al.; Chevanne et al).We extended this evaluation to ANK and TPR motif NLR proteins of Podospora, in an effort to identify no matter whether repeat size polymorphism and diversifying choice was a popular property of such repeat domains.We selected eight P.anserina NLRencoding genes displaying extremely conserved ANK and TPR motifs, and PCRamplified the repeat region from genomic DNA from 5 various wild isolates.For each 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 recommend frequent recombination among repeats within a locus, and possibly involving loci encoding the identical style of repeats, as previously reported for WDrepeats (Paoletti et al.; Chevanne et al).Subsequent, we chosen one particular ANK repeat locus and 1 TPR motif locus for which we had sequenced the highest variety of repeats (Pa__ and Pa__, respectively) andanalysed the variability from the repeats from individual loci.For every single locus, individual repeat sequences had been aligned and analysed for position beneath positive selection (see Materials and Approaches) (fig).5 positions showed signs of positive choice in the ANK repeats and three within the TPR motifs.To locate the constructive choice and polymorphic sites around the repeat domain structure, the repeats were homologymodeled to ANK and TPR domains of recognized structure.The TPR motif domain of Pa__ was modeled employing the human kinesin light chain structure (Protein Information Bank [PDB] ID EDT) as template.Within the TPR motifs, all optimistic choice web pages at the same time as the other polymorphic position mapped for the concave side in the TPR structure within 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 one particular exception, the constructive selection and polymorphic web page also mappe.