Neurotrophic factors are polypeptide hormones required for the development and maintenance of the vertebrate nervous system. The general aim of this proposal is the study of structure-function relationships in a prototypic family of neurotrophic molecules--the neurotrophins--and in their receptors, by applying novel technologies for structure determination and studies of protein-protein interactions.
Our first objective will be to determine the crystal structure of the prototypic neurotrophin nerve growth factor (NGF) bound to a minimal fragment of the binding domain of the NGF receptor tyrosine kinase TrkA. Subdomains of TrkA complexed to NGF will also be characterised biophysically to determine conformational changes arising from the interaction of these two proteins. The crystal structure of a TrkANGF complex will be used as a convergence point of rational and random strategies for the design and identification of novel neurotrophin mimetics and inhibitors. For this purpose, we will make use of software packages that use the information provided by a crystallographically determined structure to identify small molecule analogs of NGF. In addition, we will directly investigate the roles of NGF residues forming part of the complex binding interface by engineering, production and characterization of NGF molecules bearing specific mutations. Finally, we will make use of novel techniques for production and screening of combinatorial libraries of molecules displayed on filamentous phage in order to identify novel NGF analogs. These will be applied to the screening and selection of high- affinity soluble TrkA subdomains from a collection of mutagenized TrkA fragments, and of small peptide ligands to TrkA subdomains. All novel NGF mimetics and inhibitors obtained from these approaches will be functionally characterised in batteries of biochemical and biological assays.
A second objective will be to determine the solution structure of the intracellular domain of the low affinity NGF receptor p75 (p75ICD)--the so called "cell death domain"--by nuclear magnetic resonance (NMR), and to identify and characterise synthetic and endogenous peptides capable of interacting with this domain. A novel phage display based strategy will be used to identify synthetic peptides and endogenous polypeptides capable of binding to the cell death domain of p75. These ligands will be functionally characterised in receptor binding, signalling and in biological assays. Conformational changes induced in p75ICD upon peptide binding will be studied by circular dichroism and time-resolved fluorescence.
Finally, we propose to determine the three-dimensional structure of p75ICD complexed with binding peptides by x-ray crystallography as well as by NMR. Our last and third objective will be to apply site-directed mutagenesis and novel phage display technologies to explore the amenability of the neurotrophin backbone to the engineering of novel functions.
Several domains, including a metal binding domain, a glycosylphosphatidyl inositol (GPI) anchoring domain, and a heparin binding domain will be modelled and introduced in the NGF backbone. In addition, we wish to develop a high through-put method for multiple rounds of mutation and selection of NGF variable loop regions using novel technologies for random mutagenesis and phage display. Analysis of the sequences of clones selected in this way should give high-resolution information about specific chemical groups allowing activation of the TrkA receptor in different variable loop regions of NGF. The intrinsic multi-disciplinary nature of these objectives demands the convergence of the expertise and resources of different centers. The different tasks outlined in this proposal will be performed by the coordinated effort of six different laboratories in four countries of the European Union. Each center will concentrate on specific tasks in which it has acquired a distinctive competence. The expertise collected in this way can not be obtained with in any one of the countries participating in this network.
Funding SchemeCSC - Cost-sharing contracts
WC2A 3PX London