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Crystal Structure and Characterization of the SCOC Coiled Coil Domain

dc.contributor.advisorKühnel, Karin Dr.
dc.contributor.authorBehrens, Caroline Anna Julie
dc.titleCrystal Structure and Characterization of the SCOC Coiled Coil Domainde
dc.contributor.refereeThumm, Michael Prof. Dr.
dc.description.abstractengAutophagy is a conserved eukaryotic process for the degradation of cytosolic content. An expanding double layered membrane takes up part of the cytoplasm and matures to an autophagosomal vesicle. The autophagosome fuses with the lysosome, where its content is degraded. The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). Their complex is involved in autophagy regulation. Here, I present the crystal structure of the functional important human SCOC coiled coil domain (residues 78–159), which was determined at 2.8 Å resolution. SCOC forms a parallel left handed coiled coil dimer. I observed two distinct dimers in the crystal structure with a 3-molecule asymmetric unit, showing that SCOC is conformational flexible. Importantly, several of the canonical hydrophobic a/d-heptad core positions are occupied by well conserved, polar and charged residues. I assumed that these non-canonical residues might have a strong effect on the stability and oligomerization state of SCOC. The influence of the residues was investigated by characterising mutant SCOC constructs. The double core mutations E93V/K97L and N125L/N132V led to a change from dimer to either trimer or tetramer formation and the thermostabilities of these mutants were dramatically increased. Moreover, I present the formation of a stable homogeneous complex of SCOC with the coiled coil domain of FEZ1. A minimal FEZ1 region (residues 227–290) was sufficient for interaction with SCOC. Complex formation with the SCOC double core mutants was impaired, showing that dimerization of SCOC is essential for interaction with FEZ1. In addition I identified SCOC surface residue R117 as important for binding. The human pathogen Shigella flexneri escapes autophagy by a sophisticated mechanism involving the Shigella flexneri proteins VirG and IcsB–IpgA. VirG is an autotransporter protein, consisting of a signal sequence, a passenger domain (residues 52–758) and a transmembrane domain. I established a purification protocol for the VirG (52–758) under denaturing conditions. First crystallization trials of refolded VirG passenger domain yielded spherulites and microcrystalline structures. Initial studies of IcsB–IpgA indicate, that IcsB contains flexible and presumably unfolded regions, while its chaperone IpgA is well structured and
dc.contributor.coRefereeZweckstetter, Markus Prof. Dr.
dc.contributor.thirdRefereeRodnina, Marina Prof. Dr.
dc.subject.engcrystal structure SCOCde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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