Biochemical and biophysical aspects of molecular recognition and signalling by neurotrophins

Abstract

Neurotrophins are members of a family of structurally and functionally related neurotrophic factors that control the development and maintenance of the nervous system. There are currently 5 members which make up this family: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4) and neurotrophin-6 (NT-6), which so far is only described in fish. Neurotrophin-mediated effects, (e.g. cell survival and differentiation) are caused by ligand specific binding to the trk family of tyrosine kinase receptors. NGF binds to TrkA, BDNF and NT 4 bind TrkB and NT-3 preferentially binds to TrkC. Due to the similarity in activities between NT-6 and NGF, the former is believed to act through a fish homologue of TrkA.

Furthermore, neurotrophins bind with equal affinities to the p75 neurotrophin receptor (p75NTR) which is related to members of the tumour necrocis factor (TNF) receptor superfamily. Members of this family were initially grouped according to similarities in their extracellular domains. However, recent sequence analysis revealed that some members also share similar intracellular domain of about 80 residues involved in apoptosis, called the "death domain" (DD).

Using site-directed mutagenesis and various biological and biochemical assays the critical domains and residues determining neurotrophin specificity towards binding and receptor activation were identified. Results defined discontinous stretches of amino acids in the primary structure of these proteins which, upon inspection of the prototypic three dimensional structure of NGF, delineated a continuous surface extending approximately parallel to the two-fold symmetry axis of the molecule. Using information from this structure-function analysis, chimaeric neurotrophins with novel properties were constructed such as a heparin-binding NGF and a multifunctional pan-neurotrophin-1(PNT-1).

To gain insight on the possible mechanisms of p75NTR signalling, the structure of the p75 intracellular domain (p75ICD) was determined using nuclear magnetic resonance(NMR) spectroscopy. The only structured region in our ICD construct was the conserved DD module which reveals a novel fold (shared only with Fas receptor) consisting of two perpendicular sets of three helices packed into a globular structure. A surface area devoid of charged residues (hydrophobic patch) in the DD indicated a potential site of interaction with downstream targets.

Using the Selectively Infective Phage (SIP) display technique, several peptides binding to the ICD were selected. A peptide (CFFRGGFFNHNPRYC) that interacted with the DD, was further studied by NMR and was found to bind to the above-mentioned hydrophobic patch. These selected peptides should provide leads to the natural targets of the p75ICD and also should prove to be useful reagents in probing the signalling mechanism of this receptor. Taken together, this study combines genetic, biochemical and biophysical approaches to understand the molecular basis of recognition and signalling by neurotrophins, and would help in the design of agonsists and antagonists that could mediate in disorders like neurodegeneration.