S3). Analysis of protein extracts from HTS assay such synchronously growing cells showed the presence of LdHAT1 protein at equal amounts in different cell cycle phases of L. donovani promastigotes (Fig. 1b). As the level of LdHAT1 found to be invariable during cell cycle, it would be interesting to study the effect of phosphorylation by the S-phase kinase on its activity. LdHAT1 was shown previously to interact with L. donovani S-phase cyclin LdCyc1 in a RXL-like Cy-motif-dependent manner by peptide competition assay (Maity et al., 2011). To further confirm the contribution of Cy-motif in the interaction, the putative Cy-motif of LdHAT1 was altered (290RRLVVRDDVV, LdHAT1ΔCy), and the mutated protein was used in the

interaction assay. As shown in Fig. 2a, the wild-type protein was found to interact with GST-LdCyc1, whereas the interaction with LdHAT1ΔCy was almost completely abolished, proving the involvement of Cy-motif during direct interaction between the proteins. The observation also confirmed the identity of an active Cy-motif in the molecule. The mutation at the putative Cdk phosphorylation site (394TPEKAPEK, LdHAT1-T394A) of the protein did not affect the interaction

(Fig. 2a), confirming further the specific involvement of Cy-motif in the binding. LdHAT1 was demonstrated to be phosphorylated in vitro by LdCyc1-CRK3 ABT-888 nmr complex (Fig. 2b) (Maity et al., 2011). As the substrate docking on the cyclin moiety was shown to be important for phosphorylation, to investigate the effect of Cy-motif of LdHAT1 on its phosphorylation, LdHAT1ΔCy was used as substrate in a kinase assay of LdCyc1-CRK3 complex. As observed, LdHAT1ΔCy was not efficiently phosphorylated by the kinase complex compared to the wild-type protein (Fig. 2c, lanes 4 and 5). As the mutation in Cy-motif of LdHAT1 was shown to disrupt its interaction with LdCyc1 (Fig. 2a),

the inhibition of the phosphorylation established the requirement of its docking through the Cy-motif on MRAIL-motif on LdCyc1 (Banerjee et al., 2003) for the phosphorylation Tolmetin on the target serine/threonine residue. LdHAT1 was also shown to contain a putative Cdk phosphorylation site on its C-terminal end. To confirm whether Thr-394 in the motif TPEK was phosphorylated by the kinase complex, the threonine residue was changed to alanine, and the mutant LdHAT1-T394A was used as substrate. As shown in Fig. 2c, the phosphorylation was completely abolished because of the mutation (lane 6), suggesting that the S-phase kinase LdCyc1-CRK3 targets Thr-394 for phosphorylation. It is interesting to note that Thr-394 is located very close to conserved catalytically critical Glu residue raising the possibility of regulation of HAT activity because of the incorporation of a phosphate group. Therefore, it is important to study the effect on the activity of LdHAT1 by phosphorylation of the Thr residue by the cell kinase. It was previously implicated that HAT1 from T.