MA, Natural Science Tripos / Biochemistry, Univ. of Cambridge
DPhil, Chemistry / Biophysics, Univ. of OxfordView Curriculum Vitae (pdf) View Bio(pdf)
School of Medicine E607
10900 Euclid Ave
Cleveland , OH 44116-4970
Protein-Protein Interactions in Cell Signaling of small GTPases, Plexin and Eph receptors
Our research focuses on protein-protein and protein-lipid interactions in cell signaling and migration in organ development, cancer, and macular degeneration. For this we characterize protein interactions and seek to determine how they form the molecular basis of mechanisms in cell signaling. For these studies, we use a range of structural biology, computational as well as biophysics tools, and we also collaborate with cell biologists. Both the structure and dynamics of signaling proteins are important for signal transduction. For example, what protein structural and dynamic features give rise to binding affinity and to the specificity that selects one protein binding partner over others? These studies will give us insight into the normal functions of the signaling proteins and how they are disrupted in diseased states. The knowledge gained will also help us in screening for small molecule agents that can be used to manipulate protein-protein interactions (and consequently signaling in cells) in a chemical biology approach. Furthermore, we develop methods that assist in the identification of interactions in protein complexes experimentally and improve their structural representation and characterization by molecular dynamics calculations. Of late our focus has expanded to include the transmembrane helices of receptors. We are interested in understanding how signals are transmitted across the cellular membrane via the receptor’s transmembrane helices and what role the protein domain - bilayer interactions play in the process.
Cell guidance receptors and their ligands
Figure Legend: Cell migration is guided by ligands that are secreted by target cells or attached to their membranes (top). The cell front of migrating cells, e.g. axonal growth cones (bottom), exhibits a number of transmembrane receptor families which bind these ligands (Semaphorins bind to Plexin and Neuroplin; Ephrin guidance cues bind to Eph receptors). Plexins also have a number of co-receptors, such as Met and ErbB2, which are involved in cancer cell migration and metastasis. We are trying to understand how the signals are transmitted across the cellular membrane and how the receptor’s intracellular domains interact with the cytoplasmic proteins that regulate cell migration.
Structure and Signaling Functions of the Plexin Transmembrane Receptor
This class of proteins receive guidance cues (such as binding of Semaphorin ligands, see Figure above) and is activated by them. The cues allow the cells to decide whether to move towards another cell, or away from it. This process of guidance, e.g. through the migration of a growth cone, is in part responsible for "wiring up" the nervous and the cardiovascular systems. The same family of receptors is also important in immunology and cancer. Plexins are unique, as they are the first example of a receptor that interacts directly with small GTPases, a family of proteins that are essential for cell migration and proliferation/survival. We have determined the structure of the Rho GTPase binding domain of several Plexins and in collaboration, also of the entire intracellular region of a Plexin. We are now characterizing how Rho GTPases influence the function of the receptor. Using the structural information we and others have acquired, we are also altering the binding affinity and specificity of plexin towards various GTPases.
Structure and Signaling Functions of the Eph-A1, -A2 and -B1 Transmembrane Receptors
Eph receptors comprise a highly abundant family of Receptor Tyrosine Kinases, also involved - similar to Plexins - in cell guidance, attachment and proliferation (see Figure, above). Although the structures of several of the cytoplasmic domains have been determined, the overall protein conformational changes that take place in signaling are not yet understood.
Role of Phosphorylation in small GTPase Signaling.
Using an in vitro protein biophysics approach we are characterizing phosphorylation patterns in regulatory and effector proteins that result from their exposure to a variety of active Ser/Thr as well as Tyr kinases. Biochemical assays put a limit on the effects that are observed on the protein’s function as a consequence of the phosphorylation. The relevant sites are then mutated and the role of the phosphorylation is verified in vivo.
- Hota PK & M Buck. Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions. Cell. Mol. Life Sci. 69:3765-805, 2012.
- Zhang L, A Polyansky & M Buck. Modeling transmembrane domain dimers/trimers of plexin receptors: implications for mechanisms of signal transmission across the membrane. PLoS ONE 10:e0121513, 2015.
- Zhang L, S Borthakur & M Buck. Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations. Biophys. J. 110:877-86, 2016.
- Muller-Greven J, S Kim, PK Hota, Y Tong, S Borthakur & M Buck. Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry. Methods Mol. Biol. 1493:89-105, 2017.
- Zhang L & M Buck. Molecular Dynamics Simulations Reveal Isoform Specific Contact Dynamics between the Plexin Rho GTPase Binding Domain (RBD) and Small Rho GTPases Rac1 and Rnd1. J Phys Chem B 121:1485-1498, 2017.
- Shi X, V Hapiak, J Zheng, J Muller-Greven, D Bowman, R Lingerak, M Buck, BC Wang & AW Smith. A role of the SAM domain in EphA2 receptor activation. Sci Rep 7:45084, 2017.