Lipid Transport within the Cell
Peripheral membrane proteins are important to intracellular trafficking and signaling and only bind to the outer portions of the membrane, i.e., the lipid head group and occasionally the upper portion of the aliphatic chains. Membrane binding is transient to allow for proper trafficking or signaling and typically involves anionic lipids attracting basic protein residues. Nonspecific interactions (with any anionic lipid) can be the main driving force for protein membrane attachment. However, some peripheral membrane proteins contain domains that are specific to certain lipids or membrane properties. Although many lipid binding domains are known, it is difficult to evaluate binding conformations and detailed function of peripheral membrane proteins because of weak to moderate membrane binding. Our goal is to focus on an oxysterol binding protein homolog of yeast (Osh4) that is involved in lipid trafficking between the plasma membrane (PM), endoplasmic reticulum (ER), and Golgi. The mechanism for lipid transport and membrane attachment is not well understood and we aim to clarify protein structure and function during lipid transport between organelles.
Initial studies in our lab have focused on the mechanism of lipid binding to Osh4 and how this protein binds to different membranes (see Figure). Based on lipid docking studies (Rogaski et al. JPCB, 2010, 114: 13562), certain regions of Osh4 have a strong affinity to anionic lipids. Extensive all-atom molecular dynamics (MD) simulations have shown that there exists a single binding conformation of Osh4 with negatively-charged membranes (Rogaski et al. JMB, 2012, 423: 847). This contains what was experimentally-proposed to be three unique membrane binding sites, but our studies suggest these sites may constitute a single binding site and their function is not necessarily unique.
The next step in this project is to systematically probe how this protein binds to PM/ER membranes at the same time. Osh4 has been suggested to form membrane contact sites that facilitate lipid transfer. Although this protein was originally thought to transport sterols such as cholesterol, more recently it appears that Osh4 is also involved in PI4P lipid transport that may be connected to the sterol transport pathway. Initially, an amphipathic a-helix from the lid to the lipid-binding mouth of this protein will be simulated to determine how lipid packing and curvature influence peptide binding. In addition, neutron diffraction studies will be performed at NIST to aid in describing peptide binding. MD simulations of the full-length Osh4 protein will also be used to probe dual membrane binding (PM/ER) and high-resolution field-cycling 31P NMR experiments at Boston College and other techniques will also probe protein membrane binding. The boarder impacts in research go beyond studies on Osh4 and will lead to an overall understanding of peripheral membrane proteins that traffic lipids and contain lipid packing/curvature sensing domains.
An important class of transmembrane proteins is the secondary active transporters that control the concentrations of certain molecules within a cell. These transporters couple the chemical gradient or movement of ions, such as H+ or Na+, with the movement of a solute and are expressed in all species from single-celled organisms to mammals. These proteins are involved in transporting many types of small molecules that are important for the cell to introduce or expel, e.g., ions, hydrophobic molecules, antibiotics, neurotransmitters, nutrients, and small peptides. Certain secondary active transporters are known to control the levels of antibiotics in bacteria and can lead to resistance of bacteria to certain drugs. Moreover, this class of proteins is extremely important in the human neurological system in that proteins control the release neurotransmitters.
Although understanding the transport mechanism for secondary active transporters (SATs) is important, there is limited structural data available from x-ray diffraction. Moreover, most proteins with known crystal structures only have structures of a single conformation. In other words, structural changes between the known protein conformations to states open to the periplasm or cytoplasm are not well understood. It is our aim to utilize molecular simulation methods to obtain such structures and completely describe the transport cycle of SATs. A method developed in our lab will be used, known as the IM-EX MS (implicit-explicit membrane simulation) (Pendse & Klauda JMB. 2010, 404: 506) that was tested on lactose permease (LacY, above). We are currently testing this on Mhp1 protein (above) with an ultimate goal to give structural biologists a way to determine protein structures of unknown conformations based on a single known state.
In addition to further developing a method to predict protein structure, we aim to study molecular-level transport of drugs and other substrates. For example, secondary active transporters that are important in drug resistant bacteria (antibiotics are pumped out of the cell fast that they enter) require specific antibiotic-protein interactions. Determining the protein residues that are important in this transport cycle may lead to the ability to design drugs that effectively kill the organism without being expelled by these proteins. Specifically, we will work with a member of the small multidrug resistant (SMR) family in collaboration with experimentalists.
The overall structure of the ocular lens resembles an onion due to its layers of lens fiber membranes. These membranes are unique in their high cholesterol composition, where the cholesterol to phospholipids ratio ranges from 1 to 4. The other components of the lens fiber membrane are lipids and proteins. Sphingomyelin (SM) and its derivatives constitute greater than half of the lipids in these membranes, with primarily phosphatidylcholine and phosphatidylethanolamines being the remaining lipids. The transparency of these membranes is crucial for clear vision, since an opaque lens is the cause of cataracts. The concentration of cholesterol in these membranes is believed to be important in the proper function and transparency of the lens. However, certain genetic mutations of the Major Intrinsic Protein or Aquaporin 0 (AQP0) typically result in a loss of transparency of the ocular lens at birth and possibly later in life. The exclusion of the mutated AQP0 from the membrane possibly results in vacuolated lens fibers and loss of lens transparency.
In this project, we will extend some initial work of AQP0 tetramers in a DMPC membrane (O’Connor & Klauda. JPCB. 2011, 115: 6455) to membrane interactions of protein in a native membrane, i.e., SM and cholesterol. The short-term goal of this project is to investigate the wild-type protein in a natural ocular lens membrane and compare this to an artificial DMPC membrane. Research with a DMPC membrane will verify that we can reproduce the structure and lipid-protein interactions from the x-ray crystal structure, but also predict the dynamics of the AQP0 tetramer embedded in a DMPC bilayer. Ocular lens models will be used to form a baseline for normal protein behavior to compare with simulations of protein mutations that lead early onset cataracts.The long-term goal of this project is to discern how genetic mutations of AQP0 influence the affinity of the protein for the fiber membrane and ultimately lead to cataracts. Molecular simulations of single-point and multiple-site mutations will aim to determine if these mutations alter the functionality of the protein (water transport), change the protein monomer or tetramer structure, or prevent the protein from locating in the lipid membrane. The previous wild-type studies will be crucial to understand what changes in protein properties can cause the disease symptoms.