Final Report Summary - INTRAMEMPROT (Mechanistic and structural insight into di-aspartyl intramembrane proteases) The proposed project aimed at gaining structural and functional insight on a family of membrane proteins from archaea, called PSHs (PreSenilin Homologues) which are either homologous or related in structure to the human protein Presenilin. Presenilin is an intramembrane aspartyl protease involved in several physiological processes and, notably, in the early onset of Alzheimer’s disease. As a possible backup, other intramembrane aspartyl proteases were added to the target list: prokaryotic PPPs (PrePilin Peptidases) and eukaryotic SPPs (Signal Peptide Peptidases). The challenges associated with the production and study of human presenilin are still too hard, and thus succeeding in the production of the archaeal counterparts, PSHs, as well as other peptidases (PPPs and SPPs) is necessary to lay the ground to functional and structural characterization.At the beginning of the project, only two PSHs (named MCMJR1 and MHJF) were purified at the Host Institution. Although activity as an intramembrane aspartyl protease was reported for MCMJR1 (Torres-Arancivia C et al., PLoS One, 2010), the quality of the purified samples was however too poor to allow structural studies. The primary objective was thus to express, purify and produce in a stable form as many PSHs as possible to pave the way to functional and structural characterization. We cloned 33 PSHs in 4 different vectors for etherologous expression in E. coli, as well as genes from the list of backup targets (PPPs and SPPs). We also designed and cloned 23 MCMJR1 mutants and 12 mutants of the substrate peptides, aiming at stabilizing the enzyme or at trapping stable enzyme-substrate intermediates. Following this we succeeded to express about 20 PSHs in E. coli, and to test their stability in 8-to-12 different detergent conditions. About 10 PSHs proved to be stable and monodisperse in a few or several detergents, thus scale up for each of these promising PSH/detergent couple was undertaken. This allowed us to undertake vast crystallization screening campaigns, both in classical and in lipidic mesophases, and to setup 2D crystallization experiments (collaboration with I. Ubarretxena-Belandia, Mount Sinai School of Medicine). Finally, we performed extensive optimization attempts of potential crystals, microcrystals and spherulites, as well as their analysis using synchrotron radiation.To summarize, inn line with the project goals, the outgoing phase has enabled the researcher to produce a large number of PSHs that proved stable enough in several detergent conditions and allowed to setup, well beyond the awaited outcomes, a vast campaign of crystallization screening. Unfortunately, no crystal hit could lead to good X-ray diffraction and structural investigation. In the meanwhile, the structure of a PSH has been reported in the literature (Li X. et al., Nature, 2013), as well as the structure of a PPP (Hu J. et al., Nature, 2011), while successful expression and production of SPPs could never be achieved.For this reason, a further scientific backup project has been designed in order to assure a scientific outcome from this IOF fellowship. Through a collaboration with NYCOMPS (the New York Consortium On Membrane Protein Structure) structural investigation of CDP-alcohol phosphotransferases was undertaken. These enzymes belong to ubiquitous families of membrane proteins, responsible for the biosynthesis of fundamental membrane lipids, among which cardiolipin, that is essential for respiratory processes in bacteria and eukaryotic mitochondria. The structure of a CDP-alcohol phosphotransferase from archaea could be determined, providing for the first time structural insight on the reaction mechanism of more then ten different and fundamental families of membrane embedded enzymes (Sciara et al., Nature Communications, 2014, in press).The know-how acquired by the fellow will contribute, as well, to enrich the range of methodological approaches available at the Return Host Institution, which studies membrane proteins by the means of different complementary techniques (microbiology, enzymology, redox measurements, EPR spectroscopy, proteomics). Because membrane protein studies are amongst the most challenging in the field of structural biology and biochemistry, these results constitute a major scientific outcome, and contribute to European excellence, both directly (published results) and indirectly (fellow’s knowhow).