Structure of the protein phosphatase PP1 interactors Sds22 and NIPP1

BACKGROUND The function of about one third of all proteins in our cells is controlled by phosphorylation by specific protein kinases. It is therefore not surprising that protein kinases are the target of many medicines. Most of these phosphorylations are reversible through dephosphorylation by a specific protein phosphatase. One of the most common protein phosphatases is PP1 (protein phosphatase 1). This enzyme is responsible for about one third of all dephosphorylations in the cell, and plays a role in many processes. The multifunctionality of PP1 is possible through its interaction with a 200 different activity and substrate determining proteins, by which PP1-holoenzymes are formed. An innovative therapeutic approach would be to focus on specific PP1-holoenzymes, for example by interference with the recruitment of specific PP1 binding partners. AIM This project is part of a cooperation between the Laboratory Biomolecular Architecture (Department of chemistry, K.U. Leuven) and the Laboratory of Biosignaling and Therapeutics (Department of Molecular cell biology, K.U. Leuven). The latter has built an expertise in the structural and functional characterization of PP1-holoenzymes. This research led to the selection of PP1-holoenzymes with a high therapeutic potential, including the Sds22/I3-PP1 complex. Studies show that this PP1-holoenzyme has an essential role in the complete of cell division. It is also known that the inactivation of PP1 by phosphorylation is essential in this event. These data identify the Sds22/I3-PP1 complex and phosphorylated PP1 as promising targets in the treatment of cancer. The aim of this project is to determine the detailed three dimensional structure of Sds22 and I3, in complex with PP1, and phosphorylated PP1. Based on this knowledge, crucial interaction sites within these proteins and structure-function relationships can be determined. On the long term, this information can be picked up by pharmaceutical companies to start a search for small organic molecules that interfere with the Sds22-PP1 and/or I3-PP1 interaction or with the inactivation of PP1 and to develop new therapeutics. In this way, this project will contribute to the development of the pharmaceutical industry in Flanders, and to the well-being and health of every human being. METHODOLOGY The structure of the proteins will be determined by X ray crystallography. This will be carried out in three steps: (1) Bacterial (co-)expression of the protein (or protein complex), purification (affinity chromatography, ion exchange chromatography, gel filtration) and quality control (mass spectrometry) (2) (Co-)crystallization with the use of crystallization robots (TECAN Freedom EVO 150/8 and Mosquito Nanodrop) and by means of the sitting drop and hanging drop vapor diffusion technology (3) Data collection (Bruker SMART 6000 diffractometer, synchrotron beamlines) and analysis (molecular replacement, SAD-phasing) RESEARCH GROUPS The first step will be performed in the Laboratory of Biosignaling and Therapeutics. The second and third step will take place in the Laboratory Biomolecular Architecture, that takes part in BioMacS, the K.U. Leuven Interfacultary Centre for Biomacromolecular Structure.status: publishe