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Laboratory of Molecular Biophysics
Laboratory Journal 2003
J.M. McDonnell

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J.M. McDonnell


Molecular Structures and Interactions in Immune Responses and Apoptosis


Postdoctoral associates:  Loraine Hewitt, Peter Teriete
Graduate students:  Rick Hibbert, Naomi Price, Giles Robertson, Nieshia Williams
Project student:  Gemma Baker


Research in my group focuses on several protein interaction networks: the IgE network, a set of proteins that control allergic and inflammatory immune responses, a multi-protein assembly involved in proximal signalling of FcepsilonRI (the high affinity receptor for IgE), and the BCL-2 network, a family of proteins that regulate programmed cell death.  Immune regulation, signal transduction and the control of apoptosis all rely on complex interaction pathways to regulate their activities.  In many cases individual molecules have multiple ligands and it is often difficult to know which interaction, or set of interactions, permit or prevent a biological process to occur.  Our goal is to define the set of molecular interactions for a given network, describe the structures of the individual molecules or complexes, understand the physical basis of how these interactions occur and then use this information to derive specific inhibitors of interactions.

In order to achieve these goals we need to understand the complete energy landscape of macromolecular recognition events.  Macromolecular interactions are complex; to fully understand how this occurs mechanistically will require a complete description of molecular structure and dynamics for both the receptor and ligand, and an understanding of the energetics and thermodynamics of the interaction, in a fully time-resolved manner throughout the lifetime of the bound complex as well as during the processes of association and dissociation.  Towards this end we are employing high-resolution structural methods (X-ray crystallography, NMR spectroscopy) to study molecular structures and dynamics in free and bound states, coupled with methods that allow insights into the dynamics processes of association and dissociation (SPR, fluorescence, NMR spectroscopy, photo-CIDNP, mass spectrometry).  We believe understanding these interactions pathways will allow an unprecedented understanding of the mechanism of molecular recognition and offer new insights to the development of inhibitors of macromolecular interactions. 
Time-resolved spectra.
 Figure 1:  Time-resolved NMR of Fyn SH3/ligand interaction.  Using selectively 15N-tryptophan labeled protein we have been able to describe an intermediate along the binding pathway of a polyproline peptide binding to the SH3 domain of Fyn.
 

Some recent highlights of our progress in these areas include characterizing binding intermediates using time-resolved NMR spectroscopy [figure 1] and a detailed description of the activation energies of the association and dissociation events between IgE and FcepsilonRI using surface plasmon resonance [figure 2].  In the area of structural biology of the IgE network, highlights from our work from the last year include the determination of the solution structure of CD23 (the low-affinity IgE receptor) [figure 3], a complete description its backbone dynamics and the structural characterization of the bound complexes with IgE and CD21.

Energy diagram.
 Energy diagram.
Figure 2:  Energy diagrams for the IgE/FcepsilonRI binding.  Analysis of the temperature dependence of association and dissociation events has provided insights into the energetic barriers in the recognition event between IgE and its high-affinity receptor FcepsilonRIalpha.  Values for deltaG, deltaH, deltaS, deltaG‡, deltaH‡ and deltaS‡ are shown for the wildtype interaction (a) and for the K117D mutant (b) of FcepsilonRIalpha.

Ribbon diagram
 Electrostatic surface
Figure 3:  Solution structure of derCD23. (a) ribbon diagram and (b) electrostatic surface

 

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