Studies on the third site of interaction, HYNE, have shown that the His and Tyr residues Erlotinib buy are important in the interaction with PP1c and it has been proposed that this motif functions as a degenerate RV F motif. More recent studies clearly showed that the region containing the HYNE motif interacts directly with the active site of PP1c with a major contribution of His and Tyr residues. This e cludes completely the possibility of a competition of binding to PP1c between the RV F and HYNE motifs and suggests that the His and Tyr residues of I2 promote the displacement of the catalytic metal ion. In the PfI2 pro tein, these two residues are conserved. Among the three binding sites of I2, the best identified and most widely found in PP1 partners is the 0 1 0 1 consensus motif, which corresponds to KTISW in PfI2.

The presence of RV F in about 25 30% of eukaryotic proteins is not a sufficient indicator in it self to classify a protein as a PP1c regulator. These observations, together with the fact that PfI2 is the shortest I2 protein identified so far, the absence of one binding site and the fundamental difference in the RV F motif raised the question of the cap acity of PfI2 to bind and to regulate PfPP1. Using wild type recombinant proteins, we showed that labeled PfPP1 was able to bind to PfI2 and vice versa. This was further validated by the use of a yeast two hybrid system that confirmed the interaction of wild type PfI2 with PfPP1c and suggested that it was strong since the mated PfI2 and PfPP1 yeast strains were able to grow under stringent conditions.

In order to e plore the contribution of PfI2 RV F and HYNE motifs for the interaction with PfPP1, two types of construc tions were used, one deleted for the Nt moiety of PfI2 and the other with a single mutation in the RV F motif. Binding was unaffected on SD LWH medium, whatever the construction tested and only one strain, carrying the PfI2 Y103A, mutant was unable to grow under the most stringent conditions. These obser vations show that there is no one, major site of inter action in PfI2 unlike Pf Inhibitor 3, for which we showed that the mutation of 16 W completely abolished its binding function. PfI3 e hibits a totally disorganized struc ture and seems to bind first to PfPP1 via the RV F groove and folds afterwards to accomplish its function.

Regarding I2, previous studies suggested a major role for the RV F motif along with secondary binding sites which should be intrinsically structured for efficient binding to PP1c. PfI2 secondary structure ana lysis predicted that the RV F motif is a part of an un structured Anacetrapib region, while the HYNE is within an heli . The role of this structure in PfI2 PfPP1c interaction was substantiated by the lack of binding of PfI2 deleted for the region containing the heli .