Research in the Thompson Laboratory
Our laboratory focuses on resolving the atomic structures of biological molecules that are necessary for all human life. Knowing what these proteins look like allows us to understand their function, how they are involved in different human diseases and how we can potentially manipulate them to enhance human life. Our primary technique is X-ray crystallography where we grow protein crystals and expose them to high intensity X-rays. The lab also utilizes a number of biophysical techniques and biological assays. In addition, we have established vital collaborations with other preeminent laboratories in our respective areas of research interest.
TGFB-Family of Proteins and their Antagonism
The TGFß-family represents one of the fundamental cellular signaling pathways in animals. The family is also one of most diverse groups and consists of over 30 individual signaling members or ligands. Ligands are secreted by cells and are involved in numerous biological processes during embryogenesis and in the adult animal. One particular member we have a strong interest in is the ligand myostatin. Myostatin is a strong negative regulator of muscle growth and disruption of myostatin signaling results in animals with massive gains in muscle. This has been observed in multiple animals, even humans. Therefore, inhibitors to myostatin with the goal of increasing muscle mass or preventing degradation might be one way to fight muscle wasting/atrophy in disease such as muscular dystrophies, cancer, cachexia and sarcopenia. Since too much myostatin results in low muscle mass, our bodies produce natural inhibitors to myostatin. A goal of our lab is to determine the structure of these inhibitors in complex with myostatin.
TGFß-family ligands like myostatin signal by bringing two receptors together (termed the Type I and Type II receptors). A schematic of the arrangement is show in the figure below where the ligand is colored green and purple. Our lab is highly interested in how ligands are blocked from interacting with their receptors through the interaction with other extracellular proteins. Dr. Thompson and colleagues have determined the structures of three complexes of the antagonist Follistatin or Fstl3 in complex with the ligand activin or myostatin. We have identified important mechanisms for how these antagonists bind ligands to block receptor binding and have made important discoveries on how they can antagonize certain ligands without neutralizing other TGFß ligands. Please see our structure gallery for some images of these complexes.
BMP Signaling and DAN-Family Mediated Antagonism
Bone morphogenetic proteins (BMPs) represent the largest subgroup of TGFß-family ligands, which ultimately function and signal through nearly identical mechanisms. BMPs, unlike Myostatin and Activin, control completely unique processes both during development and adulthood. In the developing embryo, BMPs play critical roles in defining cell fate specification, including bone, cartilage, foregut, neuronal, and numerous other cell and tissue types. As such, misregulation of BMP signaling has found to be critical in a number of different disease states, including diabetic kidney nephropathy, lung and kidney fibrosis, numerous cancer types, and even cardiovascular disease, particularly in atherosclerosis and plaque hardening. Because of their huge importance, different mechanisms have evolved to both fine-tune and regulate signaling.
In the Thompson lab, we are interested in understanding how BMP ligands are regulated through the action of a specific family of extracellular protein antagonists known as the differential screening-selected gene aberrative in neuroblastomaor DAN family of proteins. These proteins function through direct interaction with the secreted BMP ligands to inhibit their ability to signal. Furthermore, misregulation of DAN family proteins has been directly linked to several important disease states, including kidney fibrosis and cancer, directly because of their influence on BMP signaling. With this in mind, we are focused on better characterizing these protein antagonists and how they function to inhibit BMP signaling in the hopes of providing novel future opportunities for therapeutic intervention. To date, we have structurally and functionally characterized two proteins within this family, NBL1 and PRDC. With these structures, we have identified several regions within the amino acid critical for BMP binding and are beginning to uncover how these proteins function at the atomic and mechanistic level.
Apolipoproteins and HDL Particle Formation
Apolipoproteins are key structural elements of lipoproteins, (HDL and LDL) and critical mediators of lipid metabolism. Their detergent-like properties allow them to emulsify lipid or exist in a soluble lipid-free form in various states of self-association. They have important roles in maintaining lipid metabolism, but the molecular details of how they perform these functions remains unclear. One such question is how does apolipoprotein transition from a lipid free to a lipid bound state; additionally, how is this affected by oligomerization?
Unfortunately, these traits have hampered high-resolution structural studies needed to understand the biogenesis of cardioprotective high-density lipoproteins (HDLs). Recently, we derived a crystal structure of the core domain of human apolipoprotein (apo) A-IV, an HDL component and important mediator of lipid absorption. The structure at 2.4 A depicts two linearly connected 4-helix bundles participating in a helix swapping arrangement that offers a clear explanation for how the protein self associates as well as clues to the structure of its monomeric form. This also provides a logical basis for antiparallel arrangements recently described for lipid-containing particles. Furthermore, we propose a ''swinging door'' model for apoA-IV lipid association.
To further answer these questions and support our model for HDL particle formation, we will utilize a variety of biophysical and structural techniques, including X-ray crystallography, small angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), electron microscopy (EM) and circular dichroism (CD) to determine the conformation proteins adopts in a lipid-free and bound state.
Experimental Techniques used to Conduct our Research
The laboratory also utilizes a number of biophysical techniques and biological assays in our research. In order to generate protein crystals, we need to generate large quantities of purified protein. We over-express proteins from bacteria, insect cells and various mammalian cells and purify them on a number of chromatography columns.
Once single crystals are obtained, we expose them to X-rays with our in-house X-ray generator (above). From here, we will analyze the good crystals with high energy X-rays at synchrotrons such as Argonne National Laboratory's Advanced Photon Source. An example of diffraction from a protein crystal is shown below. Once this data is collected it can be combined with other data to generate a three-dimensional electron density map where the atoms of the protein can be placed to construct a model of the protein.
In addition to X-ray diffraction experiments utilize other biophysical techniques (some in collaboration):
Surface Plasmon Resonance (SPR)
Circular Dichroism (CD)
Small-angle X-ray Scattering (SAXS)
Nuclear Magnetic Resonance (NMR)
Fluorescence Polarization (FP)
Cell-based Transcriptional Assays
Isothermal Titration Calorimetry (ITC)