Structural Studies of Protein-Ligand Interactions in Neuronal Signal Transduction: From Alcohols to Phereomones
The action of small molecules at receptors and other proteins in signalling cascades leads to major changes in behavior. These small molecules act by producing a change in protein structure and dynamics that ultimate leads to changes in nueronal signalling. Our research focuses on two different classes of modulators of neuronal signal transduction, namely alcohols and pheromones. Alcohols act on a variety of receptors and other neuronal proteins, and lead to pharmacological changes that can result in alcohol intoxication and alcohol dependency. We are using structural biology methods such as X-ray crystallography and NMR spectrscopy to identify potential alcohol binding sites in these proteins, with the goal of understanding the mechanism of alcohol's actions.
In insects, such as mosquitoes, small molecules such as pheromones are the cues that trigger asggregation, mating and feeding. We are particular interested in studying the chemical cues that are used by disease vectors, such as the malaria mosquito Anopheles gambiae, to detect their human hosts. By determining how these molecules interact with the components of the olfactory receptor signalling cascade, we will be able to develop new approaches to intefere with the transmission of these terrible diseases.
Research in my lab uses NMR spectroscopy, X-ray crystallography, molecular biology and biophysical approaches to answer the fundamental questions of how mediators of signal transduction ineract with proteins of neuronal signalling pathways.
Molecular Basis of Alcohol's Action
A major part of the research in our lab is focussed on understanding the molecular mechanisms of alcohol's actions that lead to alcohol intoxication and alcohol dependency. Over the last decade there has been tremendous progress in defining the moelcular targets of alcohol and it has been shown that alcohol can bind directly to a number of different proteins, including ion-channels, kinases and scaffolding proteins. Binding of alcohol leads to changes in enzyme activity, sub-cellular localization or signal transduction. Even more exiting, it has been shown that single point mutations in ligand gated ion-channels such as the GABA-A and Glycine receptors can completely remove the alcohol sensitvity of these receptors. There is now strong evidence that many of the effects of alcohol are associated with a direct interaction between alcohol and the protein. Our goal is to use structural and biophysial methods to understand how binding of alcohol to these protein can produce changes in the protein function
We are using structural biology and biophysical methods to probe the molecular events involved in alcohol binding. In particular we use Nuclear Magnetic Resonance (NMR) spectroscopy and X-ray crystallography to investigate the binding of alcohol to proteins. Proteins currently being studied in the lab include model aclohol binding proteins (see below) and ion-channels and kinases that have been shown to be particularly sensitive to alcohol. Our approach is to use structural biology approaches to identify the key interactions in the alcohol-binding site. Then using molecular biology methods, in combination with fluorescence and calorimetry, we make point mutations to residues in the binding site to probe the relative contributions that these residues make to alcohol binding. From this, we have developed a working hyptoesis about what factors define an alcohol binding site, and this will help us to understand the nature of alcohol binding sites and guide us in our efforts to develop new apporahes to treat alcohol dependency.
This work is supported by NIH National Institute of Alcoholism and Alcohol Abuse (NIAAA)
|Left: Part of electron density map of Lush solved at 1.25 Angstrom resolution (Click for larger version Right: Structure of the Ethanol binding pocket in the Drosophila odorant binding protein, Lush. Ethanol binds into a hydrophobic pocket and makes hydrogen bond contacts to Ser52 and Thr57. These residues provide the scaffold of a high-affinity alcohol binding site.(Click for a larger version).|
How do Insects Smell: From Pheromones to Foot Odors
The insect olfactory system is a highly organized and incredily sensitive chemosensory system that is capable of detecting chemical odors present in the air at concentrations of only a few parts per billion. The olfactory system controls the behavioral repsonse to food, mating and migration signals. A key component of this olfactory system is a family of odorant binding proteins which bind to the odorant molecule and form a complex which is capable of activating an odorant receptor. Chemosensory responses to a specific odor depend on both the identity of the specific odorant receptor and also to the odorant binding protein. As a result it may be possible to develop novel methods to interfere with a specific signaling pathway as mechanism of pest control.
We are interested in understanding how these proteins can be targetd as a mechanism to control the spread of malaria, dengue fever and west nile virus. To this end we are using NMR spectroscopy and X-ray crystallography to probe how OBPs from different insects interact with pheromones and other chemical stimulti that are commonly encountered within the environment. This builds on our work with the LUSH.
Biochemical Basis for Alcohol Toxicity in the Brain
Chronic exposure to alcohol induces many changes in the brains of vertebrates. These include enhances the activity of an inhibitory neurotransmitter known as GABA and inhibition of the excitatory neurotransmitter N-methyl D-aspartate (NMDA). In what appears to be an adaptive process, long term exposure to ethanol results in increased levels of the NMDA receptors and changes in the expression levels of alcohol sensitive subunits of the GABAA receptor. As a result chronic exposure to ethanol followed by withdrawal can induce seizures and neuronal cell death that are associated with the increased levels of receptor activity once ethanol is removed. There is increasing evidence that the effects of alcohol are exerted partly through a direct interaction with the neurotransmitter receptors. Mutations in alcohol sensitive receptors such as the can significantly modify or even abolish their sensitivity to alcohol. The identification and characterization of alcohol binding sites in CNS receptors would provide targets for the development of pharmacological agents to control both alcohol toxicity and also alcohol dependency. Research in my laboratory is currently aimed at using NMR spectroscopy to determine the structure of the protein LUSH. This protein has a unique alcohol binding function in fruit flies. We are using this protein as a model to define the molecular nature of alcohol binding sites in proteins in order to try and understand the molecular basis of alcohol's action in the brains of mammals.
Development of New NMR Methods
NMR Spectroscopy is an inherently insensitive technique for studying biological macromolecules. It requires a lot of material in relatively high concentrations which is not always available. Part of my research involves developing more sensitive NMR experiments for the study of proteins, carbohydrates and nucleic acids. An example from a recent paper is shown below. (Click the small figures to see larger versions) This experiment was designed for oligosaccharides that have been labeled with C-13 acetyl groups. The experiment is very sensitive and also has the advantage that the complete stereochemistry of the sugar rings can be defined from this single experiment.
The HCmeCOH-HEHAHA experiment used to look at connections between acetyl groups and protons on the
rings of sugars. The resulting spectrum is shown below. Taken from Jones & Bendiak, J. Biomol. NMR, 1999 ( in press)
The 2D Spectrum of an acetylated oligosaccharide using the sequence above. The spectrum shows correlations from the acetyl ring CO group to all protons in the sugar ring.