Plant and fungal proteins identified as potential models for N/b5
Efforts to confirm the results of the CD studies described above via X-ray crystallographic studies of the N/b5 constructs were unfortunately not successful, prompting us to seek potential homologs that might crystallize more readily. BLAST searches using human Ncb5or residues 1-50 as the query revealed only a few other proteins that contain a homologous polypeptide, all from plants and fungi. In all cases the homologous polypeptide precedes a b5 domain having significant homology to that of Ncb5or and contains a Trp residue analogous to Trp37 of Ncb5or but no additional domains or regions (Figures S1, S3, S4) . No structural characterization has been performed for these homologues, and only two have been characterized functionally: RLF (R educed L ateral root F ormation protein) from plants (e.g., rice, Arabidopsis and glycine) and IRC21 (I ncreased R ecombination C enter 21) protein from yeast (e.g., Saccharomyces cerevisiae and S. pombe ). RLF appears to regulate lateral root formation independently of ARF7/19-mediated auxin signaling42 , whereas IRC21 functions in chromatin remodeling43 and DNA repair43-45likely through its binding to lipid and protein phosphatase.46, 47
The N-terminal regions of plant RLF proteins are approximately 130-residues in length (Figure 3A-B) and contain a sequence spanning residues 115-121 (Q115MDWLKL121) that is strikingly similar to residues 34-40 of Ncb5or which encompass Trp37 (L34MDWIRL40). The N-terminal regions of IRC21 proteins are approximately 77 residues in length, and in that respect are more like Ncb5or than are the plant RLF proteins. However, there is weaker homology for residues 61-67 (A61LDWHSL67) that correspond to residues 34-40 of Ncb5or (Figure 3A-B ). In addition, the sequence separating this signature from the b5 core is approximately 40 residues longer in IRC21 proteins than in Ncb5or and RLF, an insertion that may have arisen subsequent to divergence of the plant, fungi and animal kingdoms.48Yeast IRC21 and rice RLF were used for these comparisons (Figures 3B ). For these reasons we selected rice RLF protein for our structural studies, specifically residues 101-218, corresponding to residues 22-137 of human Ncb5or.