|Ph.D Student||Lahav Avital|
|Subject||Structural Investigations of Proteins Involved in Human|
|Department||Department of Chemistry||Supervisor||Professor Noam Adir|
|Full Thesis text|
In this research thesis, we studied two proteins that are part of one system; both may serve as a tool for understanding an important cellular process. Determination of three dimensional structures can provide the template to perform structure based drug design for the treating of human diseases caused by damage to this process.
COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat called coatomer creates a lattice on the membrane and concentrate cargo proteins inside the growing vesicle. Disassembly of the lattice occurs following a GTP hydrolysis on Arf1 which is controlled by ArfGAPs. This releases Arf1 into the cytoplasm, which in turn allows the release of coatomer from the membrane. The coatomer complex can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COPI subunits. The second consists of α-, β’- and ε-COPI subunits.
The structure of the carboxyl-terminal region of γ-COPI, referred to as the γ-COPI appendage, was determined previously and showed that it possesses a protein/protein interaction site on its platform subdomain. In mammalian γ-COPI this site most likely binds to the unstructured ArfGAP2 protein. In order to provide insight into the mechanism of COPI function, we are seeking a structural understanding of this interaction. We have successfully cloned a fusion protein containing the γ-COPI appendage protein fused to peptide segments of the ArfGAP2. We preformed several experiments which proved the binding of the ArfGAP2 peptide to the γ-COPI appendage in these proteins. We managed to collect one data set at 2.8Å resolution and solved this structure where we located a small part of the apparent ArfGAP2 peptide near the previously suspected binding site to the γ-COPI appendage of W776.
The coatomer's δ-COPI subunit is suspected to bind ArfGAP1 and in addition contains a sequence suspected to bind Arginine based signals that are important for recognizing proteins that should be retrieved to the ER.
We have successfully identified several crystallization conditions for the crystallization of the MHD of the δ-COPI subunit, and for the co-crystallization of this domain along with the ArfGAP1 peptide containing the δ-COPI binding site and with several Arginine based signal peptides. We have collected several data sets from the crystals and determined the three dimensional structure of the MHD of the δ-COPI subunit at 2.15Å.