X-ray crystallographic and biochemical studies of membrane vesicle protein coats.
Clathrin Domains and Model of Assembly Interface
My research program combines the powerful methods of X-ray crystallography and molecular biology to reveal the mystery of how self-assembling protein coats drive the formation of membrane vesicles within the cell. Recently we solved the X-ray structure of the self-assembling domain (Ybe et al., 1999). The clathrin triskelion has 3 protruding legs, each with an associated light chain subunit that regulates "non-productive" assembly of clathrin in the cytoplasm (Ybe et al., 1998). This light chain effect is reversed by accessory proteins, which interact with clathrin during the process of clathrin basket formation.
Watch a video of a model of clathrin self-assembly (from the Kirchhausen lab at Harvard):
Clathrin Proximal Leg, PDB ID#1B89
, Rendered in UCSF Chimera
The clathrin leg is a right-handed superhelix of a-helices and is unique in that its superhelix is straight compared to other a/a superhelical proteins. We discovered that the filamentous leg is made of seven repeating modules, CHCRI and CHCR7, extending from within the trimerization region of the flexible linker region near the globular N-terminal domain. We also found that the basic clathrin repeat is present in a number of non-clathrin proteins, which have been genetically implicated in vacuolar protein sorting. The key feature required for the complete formation of a clathrin lattice is the bend or "knee" in each leg. The knee produces a pucker in the triskelion, which allows a nascent lattice to curve and form the completed structure. The knee also makes other key interactions that make it possible for the clathrin cage to form. Without the bend, proximal domains would pair incorrectly with the distal portion of the leg, leading to a disrupted lattice. The central structural role of the bend in lattice formation suggests that its angle is an important part of how basket size is determined. This problem will be a major focus of my laboratory.
Non-clathrin coats (COPS)
The second major focus of my laboratory will be to elucidate the mechanism of COP coat formation. For years clathrin was thought to be the sole protein coat involved in vesicular trafficking. This changed when mutant yeast lacking the clathrin heavy chain was found to be viable. Morphological work also revealed some Golgi-associated vesicles were not clathrin coated, but instead were covered with a dense and less-regular coat (COPs). Vesicles trafficking from the Golgi to the ER are coated with COPI, as well as vesicles that move between Golgi stacks. On the other hand, vesicles moving away from the ER in opposite direction are covered by COPII proteins. Teasing out which subunits "talk to each other" is an essential step in unraveling the molecular mechanism of COP-mediated vesicle formation. My group will set out to identify stable subcomplexes to understand the pathway of coat formation and then use X-ray crystallography to get a molecular view of the problem.
There are several areas that we are interested in looking at, such as: