New insights into the interaction of centrin with Sfi1

Centrin binds to Rad4(XPC) and Sfil through the hydrophobic motif W(1)xxL(4)xxxL(8) in the opposite orientation. Rad4 has one motif, but Sfil has approximately 20 repeats, each of which interacts with a centrin molecule. To investigate the parameters involved in centrin binding, we purified a ScSfil domain containing 6 repeats complexed with either yeast centrin Cdc31 or human centrin 1. The present study was performed using mutagenesis of centrin and of Sfil residues involved in centrin binding and the stability of the centrin-centrin complexes was assessed using thermal denaturation and CD. Calcium stabilized these complexes, as indicated by the Tm increases measured by circular dichroism. The complexes, which were composed of Sfi1 variants and yeast centrin, were analysed in the presence of EDTA. The replacement of W with F within the repeat region yielded a functional repeat (Tm 45 degrees C). The replacement of W with A in two adjacent Sfil repeats reduced the thermal stability of the Sfi1-centrin complexes (40 degrees C). We analysed three HsCen1 variants that were homologous to the yeast mutants and induced cell cycle arrest during the G2/M transition. The HsCen1 variants E105K and F113L reduced the thermal stability (50 degrees C, 50 degrees C) of the ScSfi1-HsCen1 complexes; in contrast, the A109T variant exhibited no change in thermal stability relative to the wild -type (60 degrees C). Conversely to ScCdc31, there were no apparent centrin-centrin interactions with wild-type HsCen1, but they did occur for the S170D mutation that mimics PIA phosphorylation at the S170 residue. (C) 2016 Elsevier B.V. All rights reserved.