Final Report Summary - STRUCPER (Structural studies of peroxisomal machinery) Report on the evolution of the research group: This Marie Curie re-integration grant funded a postdoctoral fellow in my group and also provided some funds for research consumables. During the period covered by the Marie Curie grant (01/09/2009 to 31/8/2013) the research group grew from 1 postdoc and 1 technician to 7 postdocs, 2 PhD students and 2 Master students. The group is now well established at the Institute for Cell and Molecular Biology (IBMC) in Porto, occupying a well equipped research lab and publishing regularly. During the period covered by the Marie Curie grant we have published 7 research papers, 5 of which published in the past 2 years, including a Nature article (Vieira-Pires RS, Szollosi A, Morais-Cabral JH “The structure of the KtrAB potassium transporter” Nature (2013), 496:323-8), and 2 reviews. Importantly, the group has attracted external funding from the Portuguese Science foundation (FCT) and the NIH in the USA.Report on the research project:Peroxisomes are cellular organelles which in higher eukaryotes are involved in essential metabolic processes such as fatty-acid oxidation, ether glycerol-phospholipid biosynthesis and glyoxylate detoxification. Peroxisomes contain two very unique protein translocation machineries, one responsible for the import of soluble matrix proteins and the other for the insertion of membrane proteins. Due to their physiological importance mutations in the protein components of the machineries result in a spectrum of diseases named peroxisomal biogenesis disorders. The ultimate aim of the proposed research project was to develop a structural understanding of these peroxisomal translocation machineries by determining the structure of the cytosolic shuttling proteins and their membrane-bound receptors, such as Pex3p, Pex19p, Pex14p and Pex5p, alone and in complex with each other and the respective cargos. Machinery for membrane protein insertionCrystallization of Pex3p: We were able to generate very small crystals of Pex3P with a construct that included residues 34-373. Unfortunately, despite extensive trials we could not improve these crystals. We then generated several different constructs of Pex3p based on “sequence gazing” or by controlled proteolysis and mass spectrometry analysis. We obtained pure protein for some of these constructs and performed extensive crystallization trials at home and in the HTX lab in Grenoble. About the same time, the structure of a Pex3p fragment was reported in the literature by two different groups (EMBO J. 2010; 29:4083-93 and J Biol Chem. 2010; 285:25410-7). These papers describe the structure of a large fragment of Pex3p in complex with a peptide of Pex19p. The crystallized fragment of Pex3p is very similar to one of the fragments identified in our work. We decided to try to determine the structure of Pex3p in the unbound state since it would provide insights into a different aspect of the mechanism of membrane protein insertion. We performed crystallization trials covering more than 2000 conditions for each construct and obtained crystals in one of these conditions but they did not diffract and we were not able to improve its diffraction properties. These negative results are probably the result of a high degree of flexibility of Pex3p in the unbound state. Pex19p-cargo complexes:We first explored the conditions that favored complex formation between Pex19p and two different cargos, PMP24 and Pexp14. Expression and purification protocols for these proteins were optimized. Both cargo proteins were expressed and purified and several procedures for the assembly of the complexes were tried. The Pex19p-PMP24 and Pex19p-Pex14p complexes were shown to form but with low stability, falling apart in native gel electrophoresis or size-exclusion chromatography. Other assembly conditions were tried including co-expression of Pex19p and the cargo proteins but it was not possible to prepare a stable complex. We also explored the formation of the ternary complex Pex19p-Pex3p-cargo. We found out that, under several conditions, the addition of Pex3p to the Pex19p-cargo complex increased destabilization of the interaction between Pex19p and the cargo resulting in the release of the cargo and the formation of the Pex3p-Pex19p complex. This result could indicate that during the process of membrane protein insertion in the peroxisomal membrane the ternary complex is formed transiently. Crystal trials of the Pex19p-Pex3p did not generate any crystals.Machinery for matrix protein importPex5p-Pex14p complex: In collaboration with the group of Jorge Azevedo at IBMC we developed a purification protocol for Pex14p, an integral membrane protein. Surprisingly, Pex14p was very tolerant to all detergents tested. This contrasts with other membrane proteins purified in the lab which are only stable in a restricted number of detergents.The detergent-solubilized complex formed by Pex14p and Pex5p was shown to be very stable in a size exclusion chromatograph column. We performed crystallization trials with this complex solubilized in different detergents. Unfortunately we never managed to obtain any crystals.Pex5p-cargo: We established conditions for the formation of the Pex5p-catalase complex. The Pex5p-catalase complex was biochemically analyzed and it was concluded that only a small population of the complexed catalase contained heme and the complex showed very low catalase activity. In contrast, catalase expressed alone in the same conditions showed a clear presence of heme and was fully active. Attempts to crystallize the complex only resulted in catalase crystals. We also produced a version of the complex where a portion of the N-terminal region of Pex5p was removed. This complex was stable but, once again, no crystals were formed. An analysis of the molecular weight of the Pex5p-catalase complex by light scattering in-line with size-exclusion chromatography demonstrated that the complex is not monodisperse. The complex has a molecular weight varying between 420-120 kDa. The lack of monodispersity explains the difficulty in obtaining crystals of the complex and it also creates a major stumbling block for a detailed biochemical or biophysical characterization.Alternative project:The recurring problems in attaining the goals set in the original proposal led us to search for an alternative project. The new project is focused on the molecular mechanisms of regulation of potassium channels. The eukaryotic EAG or KCNH voltage gated potassium channels have important roles in cardiac repolarization, neuronal excitability and tumor growth. The cytoplasmic regions include PAS and cyclic nucleotide binding homology (CNBh) domains and are also molecular interfaces for calmodulin and kinases. Strikingly, the molecular details of the relationships between channels and cytosolic signaling proteins are still badly understood. The drosophila EAG channel plays a role in the regulation of motor neuron firing and is regulated by many different mechanisms including voltage and phosphorylation. Intriguingly the channel forms an apparently strong interaction with the protein kinase CaMKII. CaMKII is the Ca2+-calmodulin dependent serine/threonine kinase which is a major player in the reorganization of the post-synaptic density during learning processes. The kinase is a homo-dodecamer which is cooperatively activated by Ca2+/calmodulin. Unlike in the original project we have made great progress in this alternative research project. We have obtained crystals and determined a structure that details the interaction between the channel and the kinase. We have also evaluated the strength of the interaction and its dependence on several co-factors through calorimetry and surface plasmon resonance. Strikingly, the interaction between the channel and CaMKII is much stronger than the usual kinase/substrate interaction. This raises several: Does the kinase affect channel function by phosphorylation alone or also by the fact that it forms a complex with the channel and alters the channel structure? Does the dodecameric kinase act as clustering hub that brings together the channel and other protein molecules?We are now actively pursuing the answers to these and other questions. Importantly, this alternative project has opened a new line of research in the lab which involves not just structural biology but also characterization of the functional properties of the channel with electrophysiology and cell imaging. The new project has been pulling us towards a more integrated approach by connecting in vitro studies with in cell analysis. With the advances we have made we should be submitting a manuscript within a year time.