NeoProteomics

Expertise. Innovation. Results.

Applications


Our team has utilized hybrid structural methods to characterize protein-protein or protein-ligand interactions where high-resolution crystallography or NMR data are not fully available. The examples below demonstrate the power of covalent labeling methods for determining the structure of protein complexes in a variety of applications.

G protein coupled receptors (GPCRs) are key signaling seven-transmembrane domain receptors regulating signal transduction pathways and the associated cellular responses. They are the molecular targets of nearly 40% of current experimental and marketed drugs. The neurotransmitter serotonin (5-hydroxytryptamine) is an important GPCR-based signaling molecule regulating multiple physiological conditions. We used covalent labeling techniques along with computational modeling and other biochemical methods to develop a structured model for human serotonin receptor in the presence and absence of its antagonist GR125487. Our results were consistent with the predicted model and highlight a highly conserved motif in the ligand-binding pocket of serotonin receptors as a key contributor in ligand binding.

 
Figure 1 shows a two dimensional representation of a class A family serotonergic receptor sequence. Over 40 modification sites (colored in red) were found in the receptor through radiolytic footprinting experiments.  Using the constraints from experimental observations, homology modeling in conjunction with computational docking was used to examine the effects of ligand binding. The resulting 5-HT4R model and existing homologous GPCR crystal structures revealed excellent conservation of motifs in their binding pockets as shown in Figure 2.

Figure 1

A hybrid structural approach to analyze ligand binding by the 5-HT4 receptor. Padayatti PS, Wang L, Gupta S, Orban T, Sun W, Salom D et al (2013). Mol Cell Proteomics 12: 12591271
Figure 2

A hybrid structural approach to analyze ligand binding by the 5-HT4 receptor. Padayatti PS, Wang L, Gupta S, Orban T, Sun W, Salom D et al (2013). Mol Cell Proteomics 12: 12591271
 
 

Radiolytic protein footprinting with mass spectrometry and molecular modeling was used to provide an atomic model of how cofilin binds to cytoskeletal protein actin in solution form. The model identified key ionic and hydrophobic interactions at the binding interface, including hydrogen-bonding between His-87 of actin to Ser-89 of cofilin that may control the charge dependence of cofilin binding.


Figure Caption:

Actin and cofilin docking and interaction analysis. Top-scored G-actin/cofilin model from the computational docking method with the use of radiolytic footprinting experimental constraints. Red-colored region marks the closely interacting segments. Hydrogen bonding/salt bridging at the interface are indicated as stick models.


Three-dimensional structure of cofilin bound to monomeric actin derived by structural mass spectrometry data J.K. Kamal, S.A. Benchaar, K. Takamoto, E. Reisler, M.R. Chance; Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 79107915