Protein-Protein Interactions in Cell Signaling of small GTPases, Plexin and Eph receptors
We characterize protein-protein interactions and seek to determine how they form the basis of mechanisms in cell signaling. For this we use a range of structural biology, computational as well as biophysics tools, and we also collaborate with cell biologists. Both the protein structures and their dynamics are important for signal transduction in cells. Understanding their molecular determinants and their role; for example, what constitutes binding specificity? The studies will allow us to gain insight into the normal function of the signaling proteins and how it is disrupted in diseased states. The knowledge obtained will assist us in screening for small molecule agents that can be used to manipulate protein-protein interactions in a chemical biology approach. We also develop methods that help in the identification of interactions in protein complexes and improve structure and molecular dynamics calculations. Recently we are interested in how signals are transmitted across the cellular membrane via the receptors transmembrane helices and protein domain - bilayer interactions.
Structure and Signaling Functions of the Plexin Transmembrane Receptor
This protein receives guidance cues (such as binding of Semaphorin ligands) 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. throught 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. The plexin protein is unique, as it is the first example of a receptor that interacts directly with small GTPases. We have determined the structure of the Rho GTPase binding domain of several plexins and in collaboration also of the entire intracellular region. We are now characterising how Rho GTPases influence the Ras GAP function of the receptor. Using the structural information, 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. 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.
- Tong, Y, Chugha, P., Hota, PK., Li, M., Alviani, RS., Tempel, W., Shen, L., Park, HW & Buck, M. (2007) “Binding of Rac1, Rnd1 and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 Effector domain.” J.Biol.Chem. 282, 37215-37224.
- Bouguet-Bonnet, S. & Buck, M (2008) “Compensatory and long-range changes in ps-ns mainchain dynamics upon complex formation. 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1” J.Mol.Biol. 377, 1474-1487.
- Tong, Y., Bagheri-Hamaneh, M., Penachioni, J.Y., Hota, P.K., Kim, S., Alviani, R., Shen, L., Tempel., Tamagnone, L., Park., HW. & Buck, M. (2009) “Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor” J.Biol.Chem. 284, 35962-35972.
- Bagheri-Hamaneh, M., Zhang, L. & Buck, M. (2011) “A Direct Coupling between Global and Internal Motions in a Single Domain Protein? MD Investigation of Extreme Scenarios” Biophys. J. 101, 196-204.
- Lee, HJ, Hota, P.K, Chugha, P., Miao, H., Zhang, L, Kim, SJ, Alviani, R.S, Stetzig, L., Wang, B. & Buck, M. (2012) “Refined NMR structure of a heterodimeric SAM:SAM complex: Characterization and manipulation of the EphA2 interface leads to new cellular functions of SHIP2” Structure 20, 41-55.