Campbell McInnes

Growth factor receptors: Structure, mechanism, and drug discovery

The focus of this review is the relationship between the three-dimensional structure of ligands of the various members of the growth factor receptor tyrosine kinase superfamily and their interaction with the cognate receptor. Particular attention is given to the transforming growth factor-alpha, epidermal growth factor (EGF); nerve growth factor, neurotrophin; and insulin-like growth factor-1 (IGF-1), insulin systems since these have been extensively studied in recent years. The three receptor types, which bind these ligands, are the epidermal growth factor receptor family (erb B receptors), the neurotrophin or Trk receptor family, and IGF-1/insulin receptors, respectively, and represent three distinct members of the tyrosine kinase superfamily. For each of these, formation of the ligand-receptor complex initiates the signal transduction cascade through autophosphorylation by the intracellular tyrosine kinase domain. The extracellular portion of the receptor that contains the ligand binding domain in these systems varies significantly in organization in each case. For the EGF receptor system, ligand binding induces homo- and heterodimerization of the receptor leading to activation of the intracellular kinase. For the Trk receptor system, homodimerization of receptors has been shown to occur, although a second receptor, p75, is also required for high affinity binding of neurotrophins and for enhanced sensitivity of tyrosine kinase activation at low ligand concentrations. The IGF-1 and insulin receptors exist as covalent cross-linked dimers where each monomer is composed of two subunits. The aim of this review is also to discuss the mechanism of ligand-receptor interaction for each of these cases; however, since no structural information is yet available for the ligand-receptor complex, the discussion will largely be centered on the molecular requirements of ligand binding. As these receptors are activated through the ligand binding site on the extracellular domain, this represents a possible target for pharmacological intervention by inhibition or stimulation of this portion of the receptor. Thus from a drug design perspective, the focus of this review is to discuss progress in the development of agonists or antagonists of the ligand for these receptors.

Comparison of NMR solution structures of the receptor binding domains of Pseudomonas aeruginosa pili strains PAO, KB7, and PAK: implications for receptor binding and synthetic vaccine design.

The solution structures of peptide antigens from the receptor binding domains of Pseudomonas aeruginosa strains PAO and KB7 have been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans forms of these 17-residue disulfide-bridged peptides have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the PAO and KB7 peptides, with that previously determined (McInnes et al., 1993) and herein refined for the PAK peptide reveals a common structural motif. All three peptide structures contain a type I beta-turn in the conserved sequence Asp134-X-X-Phe137 and a type II beta-turn in the conserved sequence Pro139-X-Gly-Cys142. However, the overall folds of the three peptides differ as well as the disposition of the side chains comprising the hydrophobic pockets. The similarities and differences between the structures of the three strains which bind to a common cell surface receptor are discussed in light of their contributions to synthetic vaccine design.

Structure-based minimization of transforming growth factor-α (TGF-α) through NMR analysis of the receptor-bound ligand. Design, solution structure, and activity of TGF-α 8-50

The investigation of a N-terminally truncated human transforming growth factor-α (TGF-α; residues 8–50) has been completed to determine the contribution of the N terminus to receptor binding and activation. The deletion protein was proposed and designed through study of NMR relaxation and nuclear Overhauser enhancement data obtained from the TGF-α-epidermal growth factor (EGF) receptor complex, which indicated that the residues N-terminal to the A loop remain flexible in receptor-bound TGF-α and thus suggested their lack of involvement in receptor binding (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O’Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283–15292; McInnes, C., Hoyt, D. W., Harkins, R. N., Pagila, R. N., Debanne, M. T., O’Connor-McCourt, M., and Sykes, B. D. (1996) J. Biol. Chem. 271, 32204–32211). TGF-α 8–50 was shown to have approximately 10-fold lower affinity for the receptor than the native molecule in an assay quantifying the ability to compete with EGF for binding and to have a similar reduction in activity as indicated by a cell proliferation assay. NMR solution structural calculations on this molecule demonstrate correct formation of the three disulfide bonds of TGF-α 8–50 and have established the presence of native secondary structure in the B and C loops of the protein. However, some perturbation of the global fold with respect to the orientation of the subdomains was observed. These results suggest that although the N-terminal residues do not contribute directly to binding, they make a significant contribution in defining the conformation of the growth factor, which is required for complete binding and activity and is therefore significant in terms of producing native folding of TGF-α. They also show that information obtained from the receptor-bound ligand can be used to guide the design and minimization of TGF-α analogues. The implications of the study of TGF-α 8–50 for the design and synthesis of reductants of this growth factor are therefore discussed.

Modulation of specificity in cyclic antimicrobial peptides by amphipathicity

Amphipathicity of antimicrobial peptides is known to be a factor important for both antimicrobial as well as anti-eukaryotic activity. We have previously shown in cyclic antimicrobial peptides related to the head-to-tail cyclic decameric peptide gramicidin S, that changes in ring size can modulate amphipathicity through changes in secondary structure [1,2]. In a separate study we showed that the systematic incorporation of enantiomeric substitutions within the framework of a highly amphipathic cyclic tetradecameric peptide, GS14, resulted in disruption of structure and reduced amphipathicity relative to GS14 [3]. In both cases the reduction of peptide amphipathicity caused the dissociation of hemolytic activity from antimicrobial activity and resulted in peptides with a high specificity (therapeutic index).