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.