Differences in the secondary/tertiary structure between CLR-1 and CLR-3 are supported by CD analysis, which revealed slight differences in helical content among domains.
Our in vitro biochemical characterization (Ca2+ titration) of the isolated CLR domains suggests that Ca2+-binding affinities of CLR-1 and CLR-3 are similar.
Whereas average Kd1 showed a slight, but significant, difference between CLR-1 and CLR-3, there was no difference seen in average Kd2, suggesting that the CLR region of RyR1 and RyR3 may present slightly different Ca2+-binding properties (Table 3).
Both CLR domains showed monophasic binding curves with CLR-1 disignificantlyificantly higher binding affinity for Tb3+ than domain CLR-3 [ Kd=0.86±0.07 μM for CLR-1 compared with 3.8±0.5 μM for CLR-3 (n=3; P<0.001); Figures 7B and 7D and Table 2], supporting further the existence of a cation-binding pocket with moderate affinity within the primary structure of the CLR region of RyR1 and RyR3.
In N. crassa, CLR-1 and CLR-2 interact for the induction of specific genes (Coradetti et al. 2012).
In N. crassa, two other Zn2Cys6 family TFs CLR-1 and CLR-2 were the predominant regulators of the expression of cellulase-encoding genes (Coradetti et al. 2012).
Orthologues of clr-1 and clr-2 have been identified in many fungal species but some of the orthologues have been demonstrated to be functionally different.
This suggests that homologues of CLR-1 and CLR-2 could also play an important role in plant cell-wall degradation in other filamentous fungi species.
Instead, two other Zn2Cys6 family TFs CLR-1 and CLR-2 were found to be the predominant regulators of the expression of cellulase-encoding genes in N. crassa (Coradetti et al. 2012).
As controls, similar experiments were done using a clr-1 e1745); clr-1 e174513) reduction-of-function doublet-756 s2613place let-756 s26135) mutant allele.
Notably, induction of clr-2 is dependent on clr-1 in N. crassa.
