Molecular electron microscopy (EM) can address questions that are not accessible to any other structural technique. We strive to take full advantage of the unique opportunities provided by molecular EM to study structural aspects of fundamental biological questions.
Structural Studies of Membrane Proteins in Their Native Environment
X-ray crystallography of three-dimensional (3D) crystals of detergent-solubilized membrane proteins is a powerful approach for obtaining high-resolution structural information on membrane proteins. Ideally, however, membrane proteins should be studied in their native environment, the lipid bilayer. We therefore reconstitute membrane proteins with lipids into two-dimensional (2D) crystals, which we then study by electron crystallography. Membrane proteins we are currently focusing on include water channels, mitochondrial carrier proteins, and multidrug resistance transporters.
Understanding lipid-protein interactions: the aquaporin-0 membrane junction. Direct structural information on the position and conformation of lipids surrounding membrane proteins has been hard to obtain. Much of our knowledge derives from crystal structures of membrane proteins with specifically bound lipid molecules. Our electron crystallographic analysis of aquaporin-0 (AQP0) reconstituted with the lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) into double-layered 2D crystals at 1.9-Å resolution revealed nine DMPC molecules (see the figure). Since AQP0 has no specific lipid-binding sites and DMPC is not a native lipid, our structure shows a nonspecific mode of protein-lipid interactions.
AQP0 forms 2D crystals with many different lipids, offering an unusual opportunity to ask fundamental questions about how lipids interact with membrane proteins. By visualizing the structure of AQP0 2D crystals obtained with lipids with different acyl chains and different headgroups, we aim to learn how the bilayer structure adapts to accommodate the hydrophobic belt of membrane proteins and which lipid characteristics are most important for interactions with membrane proteins.
Structural Studies of Complexes Involved in Vesicular Transport
Eukaryotic cells have to move proteins and other cellular components between their organelles. This intracellular trafficking is mediated by transport vesicles that bud off from the membrane of the donor compartment and dock at the membrane of the target compartment, with which they fuse in a process catalyzed by SNARE (soluble NSF [N-ethylmaleimide-sensitive factor] attachment protein receptor) proteins. The individual steps in vesicular transport are catalyzed by a large collection of proteins that form elaborate complexes that, in turn, interact with each other and drive the process.
We are currently focusing on the multisubunit tethering complexes (MTCs), which play a central role in organizing the events that occur when a vesicle arrives at its target membrane. Through interactions with small GTPases, primarily those in the Rab family, as well as with vesicle coat proteins and phospholipids, MTCs mediate the initial, reversible interaction of a transport vesicle with its target membrane. In addition, MTCs interact with SNARE and SM proteins, thus coupling vesicle capture to the membrane fusion machinery.
MTCs can be subdivided into three groups: MTCs functioning in the secretory pathway (Dsl1, COG, GARP, and exocyst), MTCs of the endolysosomal pathway (HOPS and CORVET), and transport protein particle (TRAPP) complexes. MTCs in the secretory and endolysosomal pathways are Rab effectors and are thought to promote tethering by interacting with Rabs and SNAREs. In contrast, TRAPP complexes function as guanine nucleotide exchange factor for the Rab GTPase Ypt1/Rab1 and combine tethering with coat recognition. To understand how MTCs can orchestrate these diverse events and to obtain mechanistic insights into these processes, we use single-particle EM to establish the subunit organization of the various MTCs, to characterize their structural dynamics, and to visualize how they interact with Rabs, coat proteins, and SNAREs.
This work is also supported by grants from the National Institutes of Health.
As of August 24, 2012