PTEN and Ci-VSP Show Similar Phosphoinositide Binding Preferences

Abstract

The Ciona intestinalis voltage sensor containing phosphatase (Ci-VSP) is a voltage dependent phosphatidylinositol phosphatase with two main domains, the voltage sensing domain (VSD) and the phosphatase domain (PD). Ci-VSP's PD bears homology with the phosphatase domain of the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted chromosome 10). Recently, it has been proposed that the linker between the VSD and PD Ci-VSP constitute a Phosphoinositide Binding Motif (PBM). The PBM of Ci-VSP shares high homology with PTEN's N-terminal, which, in turn, is known to bind PI(4,5)P2 leading to an allosteric activation of PTEN. Similarly to the PBM of PTEN, we have found that a peptide representing the Ci-VSP's PBM (Ci-VSP240-271) binds PI(4,5)P2 significantly more strongly than other phosphatidyinositol bisphosphates. A Ci-VSP chimera created by replacing Arg 257 and Lys 258 with the amino acids found at the corresponding positions in the PTEN sequence, Gln 16 and Glu17, lead to a peptide that showed significantly reduced binding to PI(4,5)P2. While Ci-VSP240-271 exhibited a mixture of random and a-helical secondary structural elements, it was found that the chimeric Ci-VSP showed an increased b-sheet content. Molecular dynamics simulations were performed using the package NAMD and showed that the peptide does not form a helical structure and its charged residues interact in a pairwise fashion with PI(4,5)P2. Those pairs are form by R245 and R246, K252 and R253, and R254 and R257. Experimentally, the binding of the PBM has a great influence on the rate of return of the sensing charges. However, in a deletion mutant lacking the PD we observed that the PBM alone is not enough to limit the rate of return of the sensing currents. These observations strongly suggest that the binding of the PBM might be stabilized by the PD.