Research

Our research goal is to understand how protein domains transduce signals from biological membranes. Our laboratory employs biophysical approaches - including high field nuclear magnetic resonance spectroscopy, isothermal calorimetry, circular dichroism, computer modeling, liposome-binding assays, fluorescence spectroscopy and surface plasmon resonance spectroscopy - to determine protein:lipid interfaces, ligand binding pockets and membrane insertion of protein domains from the molecular to the atomic resolution. We validate our functional and structural approaches by using normal and disease-associated cell lines. With these tools we can establish how protein-ligand interactions control the function of proteins by modulating their subcellular localization.

Role of Disabled-2 in Sulfatide-mediated Platelet Aggregation

Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin a_IIbb₃ receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin a_IIbb₃ receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively.

Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, we identified the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains.

Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms. This project is in collaboration with Carla Finkielstein and Anne Brown (Virginia Tech).

Role of Modular Proteins in Endosomal Protein Trafficing

HSQC spectrum of 15N-labeled Tollip TBD in the absence (left) and presence (right) of the Tom1 GAT domain.

Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. We focus on structural studies of two adaptor proteins, the Toll-interacting protein (Tollip) and Tom1. These proteins form a complex named alternative ESCRT-0 complex that is responsible for the transportation of cargo at the endosomal compartments. We have previously demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides and that this association is critical for membrane targeting of the protein. Remarkably, we found that ubiquitin modulates Tollip’s lipid binding. We have observed an ubiquitin dose‐dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to hypothesize that the Tollip C2 domain is a novel ubiquitin‐binding domain. Also, we have obtained detailed structural information about how Tom1 and Tollip form a complex. Their association is mediated by the TBD and C2 domains (in Tollip) and the GAT domain (in Tom1). Overall, our findings will provide the structural basis to understand how cargo is transported by the Tollip-Tom1 complex in endosomal compartments. This project is in collaboration with Carla Finkielstein (Biological Sciences, Virginia Tech), Anne Brown (University Libraries, Virginia Tech), Jeff Ellena (University of Virginia), Samppa Ryhänen, Elina Ikonen, and Heljä Lång (New Children Hospital, University of Helsinki, Finland)

Role of Adaptor Proteins in Autophagy

We are interested in understanding the function of phosphatidylinositol 3-phosphate (PtdIns3P)-binding adaptor proteins in autophagy. Phafin2 is a PtdIns3P binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns3P is mediated by both its PH and FYVE domains. We found that Phafin2 is a moderately elongated monomer of ~28 kDa with an intensity-average hydrodynamic diameter of ~ 7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits a / structure and predicts ~40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding-unfolding curves, obtained using urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ~4 kcal mol-1. PtdIns3P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. With these studies, we intend to determine the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns3P-binding domains of the protein in endomembrane compartments. This project is in collaboration with Rana Ashkar (Virginia Tech).