BMC Genomics 2008, 9:308 http://www.biomedcentral.com/1471-2164/9/308 RFHAiMgWuMr-UeHL2MaMndpUrobflildeocmoaminpsarison E-Values between the HMM-HMM profile comparison E-Values between the RAWUL and Ubl domains. The numbers correspond to HMM-HMM profile comparisons E-values from global pro- file search results [49] that connect independently each fam- ily with the others. The arrows indicate the profile search direction. domain is more conserved inside the WDR48 family than in PRC1 Ring family. Nevertheless, the homology of the WDR48 RAWUL domain with the UBL family is statisti- cally supported, when using as intermediate the RAWUL HMM profile of PRC1 Ring family. This result defines a new conserved domain present in the PRC1 Ring finger and WDR48 families. In all of those pro- teins the domain is always observed in a C-terminal loca- tion (Figure 3), suggesting that this is somehow important for its function. Statistically significant sequence similarity offered by pro- file-profile comparisons linked this domain with ubiqui- tin superfamily (Figure 1 and 2). Ubiquitin (Ubq) is a highly conserved 76 amino acid residue protein found in all eukaryotic cells and whose sequence is extremely well conserved from protozoa to vertebrates [53]. The overall topology of the Ubq-like fold is β1, β2, α1, β3, β4, α2, β5 (where β indicates beta strand and α alpha helix); how- ever, there may be deviations from this common core [54]. Although those E-values are significant per se, we per- formed three additional analyses. First, secondary struc- ture predictions were performed independently for the conserved C-terminal regions of PRC1 Ring finger and WDR48-p80 families [55] (Figure 1). These predictions showed good agreement with the Ubq and Ubq-like struc- tures (Figure 1). Second, to investigate whether fold recog- nition analysis generated consistent results, we submitted the Ubq-like domain of the human Bmi-1 protein (Uni- prot-id: BMI1_HUMAN, residues 131 to 226) to an inde- pendent fold assignment software (mGenThreader [56]). mGenthreader consistently recognised only Ubq-like pro- teins as possible templates (8 out of the 10 proposed hits are Ubq-like folds). This consistency in the threading out- put additionally supports the relationship between Ubq and the conserved PRC1 Ring C-terminal domain. Third, we generated a structural model (see Additional file 1) of the human Bmi-1 putative Ubq-like domain based on alignments including ubiquitin superfamily sequences of known structure (Figure 1). The model was evaluated using statistical mean force potential and found to have a Z-score of -6.5, in the upper range of good model for this length [57], strengthening the assignment of the Ubq fold. Despite the low sequence identity between this domain and the Ubq (below 20%), the hydrophobic core and key secondary structural elements of the Ubq fold are con- served (Figure 1) [54]. However, the PRC1 Ring finger family displays a higher degree of sequence variability in the C-terminal Ubq-like domain than the WDR48-p80 family. The domain secondary structural elements are rich in charged residues and are separated by loops of variable length (Figure 1 and Additional file 2), which are likely to be the reasons why this domain escaped detection until now. The fact that the domain shows insertions of varia- ble length is not in contradiction with a stable fold [58]; even more, small folds, such as Ubiquitin [54], Immu- noglobulin [59] or SBDS [60], share a high degree of structural conservation in a scenario of high sequence divergence and long loop insertions. The most characteristic features of this domain is the α2 that is a bit larger in its C-terminus compared to classical Ubq-like proteins and contains a conserved pair of Thr/ Leu that might have a structural role in capping the alpha helix (Figure 1). Preliminary structural characterization of the C-terminal region of Ring1B showed that this domain folds independently and is a combination of alpha-helix and beta-sheet secondary structures [61], which is in agreement with our analysis. In summary, considering the profile-profile comparisons E-values (Figure 2), secondary structure predictions (Fig- ure 1), fold assignment detection and the results of the homology model building and evaluation (see Additional file 1), we are confident that this domain is a new member of the Ubq superfamily. Since we found this new Ubq-like domain associated to two different domain architectures, Ring-finger domain (in PRC1 Ring family) and WD40 repeats (in WDR48-p80 family), we named it RAWUL (Ring-finger And WD40 associated Ubiquitin-Like) domain. Page 4 of 11 (page number not for citation purposes)