Inhibition or lack of axon guidance affects neuronal cell motility and positional maintenance, resulting in the failure to regenerate neurona! connections after injury. Defective guidance is also associated with diseases ranging from Alzheimers to cancer. The long term objective of our research is to determine the biophysical basis of signal transduction mechanisms involving axon guidance cue receptors and small GTPases. The Plexin-B1 transmembrane protein is the first example of a receptor that interacts directly with small GTPase, Rac1. The major goal of the proposed project is to provide a detailed structural and thermodynamic characterization for the basis of the plexin-Rac1 interaction and to understand its role in plexin mediated signal transduction. Our hypothesis is that the propensity to populate different conformational states is already a characteristic of the Rac1 Binding Domain, and that such regulatory switching behavior will be apparent in this plexin domain by itself, if not upon its interaction with the GTPase or in response to other perturbations.
Specific aims : 1. Determination of the structure of the Rac1 binding domain of plexin-B1 and characterization of its dimerization interface and of the putative Rac1 binding switch. Nuclear Magnetic Resonance (NMR) spectroscopy will be used for structure determination of a monomeric form of plexin, and will be utilized to map the plexin dimerization interface of the wild type protein. 2. Characterization of the plexin Rac1 binding domain - GTPase interface, also in a non-binding mutant, and determination of the structure of the protein plexin-Rac1 complex. 3. Thermodynamic perturbations caused by plexin:GTPase complexation will be quantified and the relationship between stability and binding affinity of the structures involved will be determined. Global thermodynamic perturbations will be followed by fluorescence and circular dichroism spectroscopy, site specific changes by amide hydrogen exchange in conjunction with NMR spectroscopy. Significance: These studies will determine the biophysical basis for a signaling mechanism involving a small GTPase - protein interaction and will provide deeper insights into other interactions that involve small GTPases in signaling events.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM073071-01
Application #
6859248
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Flicker, Paula F
Project Start
2005-02-01
Project End
2010-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
1
Fiscal Year
2005
Total Cost
$260,100
Indirect Cost
Name
Case Western Reserve University
Department
Physiology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Zhang, Liqun; Sodt, Alexander J; Venable, Richard M et al. (2013) Prediction, refinement, and persistency of transmembrane helix dimers in lipid bilayers using implicit and explicit solvent/lipid representations: microsecond molecular dynamics simulations of ErbB1/B2 and EphA1. Proteins 81:365-76
Cao, Shufen; Mao, Xi'an; Liu, Deli et al. (2013) Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase. Biomol NMR Assign 7:121-8
Seerapu, Himabindu Reddy; Borthakur, Susmita; Kong, Nathan et al. (2013) The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover. FEBS Lett 587:3392-9
Lee, Hyeong J; Hota, Prasanta K; Chugha, Preeti et al. (2012) NMR structure of a heterodimeric SAM:SAM complex: characterization and manipulation of EphA2 binding reveal new cellular functions of SHIP2. Structure 20:41-55
Zhang, Liqun; Bouguet-Bonnet, Sabine; Buck, Matthias (2012) Combining NMR and molecular dynamics studies for insights into the allostery of small GTPase-protein interactions. Methods Mol Biol 796:235-59
Hota, Prasanta K; Buck, Matthias (2012) 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
Hamaneh, Mehdi Bagheri; Zhang, Liqun; Buck, Matthias (2011) A direct coupling between global and internal motions in a single domain protein? MD investigation of extreme scenarios. Biophys J 101:196-204
Wang, Hui; Hota, Prasanta K; Tong, Yufeng et al. (2011) Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs). J Biol Chem 286:26093-106
Zerbetto, Mirco; Buck, Matthias; Meirovitch, Eva et al. (2011) Integrated computational approach to the analysis of NMR relaxation in proteins: application to ps-ns main chain 15N-1H and global dynamics of the Rho GTPase binding domain of plexin-B1. J Phys Chem B 115:376-88
Balakrishnan, Asha; Penachioni, Junia Y; Lamba, Simona et al. (2009) Molecular profiling of the ""plexinome"" in melanoma and pancreatic cancer. Hum Mutat 30:1167-74

Showing the most recent 10 out of 18 publications