The TGFβ superfamily consists of over 30 unique signaling ligands which control cellular functions ranging from reproduction and development to homeostasis and wound repair. These ligands can be further subdivided into three classes: the TGFβs, for which the family is named, the bone morphogenetic proteins (BMPs), and the activins, which play roles in reproduction and muscle development. Family proteins are synthesized (A) as longer precursors containing a large N-terminal prodomain (~250 residues) and a smaller C-terminal growth factor domain (~110 residues) which are proteolytically cleaved (B) during processing but can remain non-covalently associated during trafficking and post-secretion (C). A single ligand dimer can assemble a signaling complex (D) by association with two type II receptors and two type I receptors. When assembled as such, the constitutively active type II receptors can phosphorylate and activate the type I receptors (E), and in turn the type I receptors can phosphorylate and activate intracellular Smads (F) which then translocate to the nucleus and induce or repress the transcription of target genes. The 8 Smad proteins are divided into three subclasses: receptor-regulated Smads (R-Smads) 1, 2, 3, 5, 9, common partner Smad (Co-Smad) 4, and inhibitory Smads (I-Smads) 6 & 7. When TGFβ signaling is active, heterotrimeric complexes of two R-Smads and one Co-Smad accumulate in the nucleus and proceed modulate transcriptional activity directly, by binding DNA, or indirectly by influencing the action of other transcription factors.
Questions of activation, regulation, and specificity are inherent when studying TGFβ family ligands. Distinct extra- and intracellular mechanisms have evolved to regulate each class at several levels, providing exquisite control over structurally related signaling molecules. Within the family, there are only five type II receptors (TβR2, ActRIIA, ActRIIB, BMPR2, AMHR2) and seven type I receptors (ALK1-7) available. Furthermore, the receptor composition of the signaling assembly ligand dependent: this creates a receptor bottleneck for the more numerous ligands. Many classes of ligands are exclusive to their type II receptor; TGFβs, for example, are exclusive for TGFBR2 and AMH is mutually exclusive with AMHR2. Since its inception, the long-term objective of our laboratory – funded by multiple R01 awards – has been to provide a molecular understanding of the mechanisms of TGFβ family signaling and its regulation. In the investigation of these ligands, the application of structural techniques coupled with biophysical studies has provided foundational knowledge of the molecular mechanisms governing their ubiquitous, high impact pathways. In response, there has been a surge of interest from the drug development industry to modulate points along these pathways for therapeutic gain.
Our lab currently concentrates on the structural determination and biophysical examination of proteins within the TGFβ superfamily. We are most interested in questions concerning extracellular modulation of BMP and activin-class signaling and how these mechanisms, once understood, might be manipulated to enhance human life. Using a combination of X-ray crystallography and binding analysis coupled with in vitro cellular assays, the objective of our laboratory is to define the molecular mechanisms of ligand-receptor interactions incorporated to differentiate signaling. Furthermore, our laboratory is characterizing the interactions of extracellular antagonists, which neutralize ligands by blocking ligand-receptor interactions. Similarly, we aim to understand how the N-terminal prodomain of certain ligands renders the growth factor latent or otherwise regulates it, with a focus on deciphering the molecular mechanisms of activation. Most recently, we have begun to incorporate outlier members of the TGFβ family with therapeutically relevant reproductive roles, such as AMH, into our investigative wheelhouse. In addition, the literature has shown that heterodimeric ligands can form and are biologically relevant in certain cases, even more so than the cognate homodimeric ligands. Thus, our laboratory is investigating the structure, function, and synthesis of ligand heterodimers. In a disparate project, we have characterized the structure and function of apolipoproteins with the intent to understand how they transition from a lipid free state to a lipid bound state in the biogenesis of lipoprotein particles.
Structure-Function Studies of Ligand-Receptor Interactions
To better understand the general mechanisms of TGFβ signaling it is important to understand, at the molecular level, how ligands bind and interact with their signaling receptors. This information illuminates how certain ligands are able to signal through specific combinations of type I and type II receptors. Within the three ligand classes, differences in receptor affinity exist such that BMP ligands bind type I receptors with high-affinity while both TGFβ and activin ligands display low affinity for type I receptors. Different ligand classes utilize different receptor combinations and can be more or less promiscuous. For example, while all TGFβ class ligands signal using ALK5, activin class ligands are capable of signaling through ALK4, ALK5 and ALK7. Structural studies have revealed differences in the mechanisms for TGFβ and BMP ligand assembly of type I and type II receptors. For TGFβ class ligands, a cooperative assembly paradigm is utilized where the type I and type II receptors directly interact in the presence of the ligand, in contrast to BMP class ligands, where the receptors bind independently. While structures of TGFβ and BMP ligands bound to their receptors are available, we have limited information for how Activin class members assemble a ternary signaling complex.
Mullerian Inhibiting Substance (MIS) or Anti-Mullerian Hormone (AMH), originally identified for its role in male sex differentiation during development, has emerged as a significant molecule in female reproduction. Gonadal AMH plays a major role in regulating follicle development and blood serum levels are used as a measure of ovarian reserve and as a diagnostic tool. Mutations in AMH are associated with both male and female reproductive disorders, including Persistent Mullerian Duct Syndrome (PMDS) in males and Polycystic Ovary Syndrome (PCOS) in females. As a member of the TGFβ family, AMH signals through a type I and type II receptor. Uniquely, AMH signals through its own type II receptor AMHRII, but utilizes the type I receptor ALK2, which is shared by multiple ligands. While previous studies have detailed TGFβ family ligand interactions, how AMH interacts at the molecular level with AMHRII and ALK2 is unknown. Our lab seeks to understand the unique structural characteristics, how AMH complexes with its cognate receptors and how AMH signaling is generated using these receptors. A more complete understanding of AMH can provide a platform for developing therapeutics that modify AMH activity with the potential for future application in reproductive therapies.
Extracellular Ligand Modulators
Due to the potency of TGFβ ligands and the wide variety of biological targets they regulate, the modulation of TGFβ signaling is of key importance. Ligands are regulated by over 20 structurally diverse extracellular protein antagonists. Over the past 15 years, our lab has made significant contributions towards understanding the diversity of the extracellular protein antagonist and how they neutralize ligands. Our initial efforts were focused on the follistatin family where we solved a battery of follistatin-ligand structures, including follistatin:activin A, FSTL3:activin A, follistatin:GDF8, FSTL3:GDF8, and follistatin:GDF11. These studies emphasized the complexity of ligand-antagonist interactions, including conformational plasticity to accommodate differences at the ligand-antagonist interface. Over the years, we have extended our studies to include the DAN-family and WFIKKN-family antagonists. Similar to follistatin, WFIKKN is a multi-domain antagonist, however it has much more limited specificity – it specifically antagonizes GDF8 and GDF11. In contrast, the DAN-family consists of a small, single domain antagonists that neutralize ligands of the BMP subclass.
Activin-class Antagonism by Follistatin and Structurally-related Proteins
Protein antagonists consist of varying domain architectures and inhibit ligand-receptor interaction through different binding mechanisms. The majority function by blocking both the two type I and two type II receptor binding sites to prevent receptor assembly and signaling. In 2005, Dr. Thompson solved the second ever published antagonist-ligand complex between follistatin and activin A. Follistatin is a multidomain protein containing an N-terminal Domain (ND) and three tandem follistatin domains (FSDs), two molecules of which bind the activin A dimer in a ring-like inhibitory structure. The ND of each follistatin occupies the type I receptor binding site with FSD1 and FSD2 covering the type II receptor site. The two follistatin molecules interact in a head-to-tail mechanism where the ND of one Follistatin interacts with the FSD3 of the neighboring Follistatin.
Structures from our lab of follistatin in complex with other ligands have highlighted a conformational selection model where both the antagonist and ligand adopt a preferred conformation for binding. The ND of follistatin can adopt different conformations to accommodate many ligands. This mechanism provides the basis for the promiscuity of follistatins. We have a particular interest in inhibition of GDF8, also known as myostatin. Myostatin is a strong negative regulator of muscle growth – disruption of signaling results in animals with massive gains in muscle, an outcome observed in many species, including humans. The development of potent and selective inhibitors to myostatin with the goal of increasing muscle mass and preventing degradation might be one way to fight muscle wasting/atrophy in muscular dystrophies, cancer, cachexia, and sarcopenia. The body’s natural antagonists, such as follistatin and FTSL3, are of significant therapeutic relevance.
BMP-class Antagonism by Differential Screening-selected Gene in Neuroblastoma (DAN) family Proteins
The DAN-family represents the largest single family of extracellular antagonists with 7 total members. Each protein consists of a core DAN domain that is composed of four conserved disulfide bonds, adopting a growth-factor like shape. Our initial work showed that DAN-family members, specifically Gremlins, formed stable non-disulfide bonded dimers. When we solved the structure of gremlin-2 in complex with the ligand, we found that DAN antagonists use a completely novel binding mode compared to other known antagonists. This represents a foundational set of bound and unbound antagonist structures, giving insight into the molecular transitions that occur upon ligand binding. Currently, we are studying divergent DAN family members, SOST and SOSTDC1.
Structure-function studies of Heterodimers
Most all TGFβ family ligands form disulfide-bonded dimers. Over the past three decades, the field has primarily focused on homodimers formed from 2 identical subunits. However, emerging evidence indicates that heterodimers play a vital role in signaling. In collaboration with Martin Matzuk (Baylor College of Medicine), we showed that GDF9:BMP15 heterodimers were essential for folliculogenesis. Further studies have suggested that only heterodimers signal during development. Considering that there are over 30 distinct ligand monomers, the number of potential heterodimers is over 500, providing a vast new dimension to TGFβ signaling. We have initiated projects in our laboratory that will address the function, formation, and signaling of TGFβ heterodimers.
Prodomain-Ligand Interactions
Ligands within the TGFβ family are synthesized with a prodomain that facilitates proper assembly of the TGFβ dimer before cleavage by the protease furin. Evidence suggests that most ligands remain noncovalently associated with their prodomains. In a number of cases, this interaction does not inhibit signaling – in others it can even potentiate ligand signaling. Conversely, the prodomain can also render the ligand latent, keeping it trapped in a non-productive prodomain-ligand complex or procomplex.
We aim to further characterize and illuminate the role that prodomains have on ligand formation and signaling.
Experimental Techniques
“structure without function is a corpse; function without structure is a ghost” -Vogel and Wainwright, 1969
While our research program includes various and diverse research practices, the backbone and basis of our research is structural biology. While historically focused on X-ray crystallography (XRC), the lab is currently incorporating single particle Cryogenic Electron Microscopy (Cryo-EM) into many of our projects. We consider the structural information we generate only a starting point, and we approach functional validation with equal effort. our typical studies include cell-based endpoint signaling assays and protein-based binding measurement using Surface Plasmon Resonance (SPR). We are currently expanding our structural toolset to include more advanced computational techniques and modeling, meanwhile we are incorporating kinetic signaling assays with the through a collaboration with the Elowitz lab at Caltech.
In addition to our core capabilities, we also utilize these other biophysical techniques (some in collaboration):
Circular Dichroism (CD)
Small-angle X-ray Scattering (SAXS)
Fluorescence Polarization (FP)
Analytical Ultracentrifugation (AUC) Cyro-Electron Microscopy (Cryo-EM)
Funding
R01HD105818 - Structure-function analysis of Mullerian Inhibiting Substance (MIS)
R01AG072087 - Regulation of GDF11 by extracellular mechanisms
R35GM134923 - Structural/functional characterization of TGFβ superfamily signaling and regulation
S10OD030388 - Glacios 200 kV cryogenic transmission electron microscope (cryo-TEM)
