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J Murray-Rust

Publications and source records attributed to J Murray-Rust.

16 recordsLinked to original sources
PubMed1996

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding.

Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was approximately 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was approximately 10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.

Amino Acid Sequence↗
PubMed1995

Functional analysis of mutant neurotrophins deficient in low-affinity binding reveals a role for p75LNGFR in NT-4 signalling.

The neurotrophins mediate their effects through binding to two classes of receptors, a tyrosine kinase receptor, member of the Trk family, and the low-affinity neurotrophin receptor, p75LNGFR, of as yet undefined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-directed mutagenesis, we have identified positively charged surfaces in BDNF, NT-3 and NT-4 that mediate binding to p75LNGFR. Arg31 and His33 in NT-3, and Arg34 and Arg36 in NT-4, located in an exposed hairpin loop, were found to be essential for binding to p75LNGFR. In BDNF, however, positively charged residues critical for p75LNGFR binding (Lys95, Lys96 and Arg97) were found in a spatially close but distinct loop region. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials revealed similar clusters of positively charged residues in each neurotrophin but with differences in their precise spatial locations. Disruption of this positively charged interface abolished binding to p75LNGFR but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity binding in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75LNGFR and TrkB, suggesting a role for p75LNGFR in regulating biological responsiveness to NT-4. These findings reveal a possible mechanism of ligand discrimination by p75LNGFR and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family members.

3T3 Cells↗
PubMed1994

Nerve growth factor: structure/function relationships.

Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted beta-hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell-surface receptor types expressed in a variety of neuronal and non-neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site-directed mutagenesis, and this information can be related to their location in the 3-dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both promoters in the dimer.

Amino Acid Sequence↗
PubMed1993

Topological similarities in TGF-beta 2, PDGF-BB and NGF define a superfamily of polypeptide growth factors.

BACKGROUND: The development of functional diversity through gene duplication and subsequent divergent evolution can give rise to proteins that have little or no sequence similarity, but retain similar topologies. RESULTS: The crystal structures of nerve growth factor, transforming growth factor-beta 2 and platelet-derived growth factor-BB show that all three are based on a cystine-knot plus beta-strands topology. There is very little sequence identity between the three proteins and the relationship between the structures had not been deduced from sequence comparisons. Each growth factor is usually active as a dimer; each exists as a dimer in the crystal, but the relative orientations of the protomers are different in each case. CONCLUSION: The structural motif of disulphide bonds and hydrogen-bonded beta-strands unexpectedly found in these three growth factors acts as a stable framework for elaboration of loops of low sequence similarity that contain the specificity for receptor interaction.

Amino Acid Sequence↗
PubMed1993

An extended surface of binding to Trk tyrosine kinase receptors in NGF and BDNF allows the engineering of a multifunctional pan-neurotrophin.

Neurotrophin-mediated cell survival and differentiation of vertebrate neurons is caused by ligand-specific binding to the Trk family of tyrosine kinase receptors. However, sites in the neurotrophins responsible for the binding to Trk receptors and the mechanisms whereby this interaction results in receptor activation and biological activity are unknown. Here we show that in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), discontinuous stretches of amino acid residues group together on one side of the neurotrophin dimer forming a continuous surface responsible for binding to and activation of TrkA and TrkB receptors. Two symmetrical surfaces are formed along the two-fold axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. Mutated neurotrophins inducing similar levels of receptor phosphorylation showed different biological activities, suggesting that structural differences in a ligand may result in dissimilar responses in a given tyrosine kinase receptor. Our results allowed us to combine structural elements from NGF, BDNF and neurotrophin-3 to engineer a pan-neurotrophin that efficiently activates all Trk receptors and displays multiple neurotrophic specificities.

Amino Acid Sequence↗
PubMed1993

Nerve growth factor revisited.

Recent studies on nerve growth factor have revealed important new insights into the structure, function and evolution of this prototypical neurotrophic factor. Some of its features are (1) it has a unique three-dimensional fold that has since been found in two other growth factors, (2) it uses the trk proto-oncogene product, which has a tyrosine kinase, as a receptor and (3) it shares homology with at least three other factors, now collectively called neurotrophins, which have a spectrum of target cells.

Amino Acid Sequence↗
PubMed1992

Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product.

Nerve growth factor (NGF), like many other growth factors and hormones, binds to two different receptor molecules on responsive cells. The product of the proto-oncogene trk, p140trk, is a tyrosine kinase receptor that has been identified as a signal-transducing receptor for NGF, while the role of the low affinity NGF receptor, p75NGFR, in signal transduction is less clear. The crystal structure of NGF has recently been determined, although structures involved in receptor binding and biological activity are unknown. Here we show that Lys-32, Lys-34, and Lys-95 form a positively charged interface involved in binding to p75NGFR. Simultaneous modification of Lys-32 with either of the two other lysines resulted in loss of binding to p75NGFR. Despite the lack of binding to p75NGFR, these mutants retained binding to p140trk and biological activity, demonstrating a functional dissociation between the two NGF receptors.

Amino Acid Sequence↗
PubMed1992

Structure and evolution of insulins: implications for receptor binding.

Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common 'insulin fold' is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.

Binding Sites↗
PubMed1991

New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor.

Nerve growth factor (NGF) is a member of an expanding family of neurotrophic factors (including brain-derived neurotrophic factor and the neurotrophins) that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous systems. Its biological effects are mediated by a high-affinity ligand-receptor interaction and a tyrosine kinase signalling pathway. A potential use for NGF and its relatives in the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease requires an understanding of the structure-function relationships of NGF. NGF is a dimeric molecule, with 118 amino acids per protomer. We report the crystal structure of the murine NGF dimer at 2.3-A resolution, which reveals a novel protomer structure consisting of three antiparallel pairs of beta strands, together forming a flat surface. Two subunits associate through this surface, thus burying a total of 2,332 A. Four loop regions, which contain many of the variable residues observed between different NGF-related molecules, may determine the different receptor specificities. A clustering of positively charged side chains may provide a complementary interaction with the acidic low-affinity NGF receptor. The structure provides a model for rational design of analogues of NGF and its relatives and for testing the NGF-receptor recognition determinants critical for signal transduction.

Amino Acid Sequence↗
PubMed1990

X-ray crystallographic studies on murine nerve growth factor.

The largest and best characterised family of neurotrophic growth factors is that of nerve growth factor (NGF) and its relatives. In order to understand the relation of structure and function, we have undertaken X-ray analyses of murine NGF. The active component beta-NGF crystallises as hexagonal bipyramids that give good X-ray diffraction data using a synchrotron to 2.3A resolution. We have prepared several heavy atom derivatives that are being used in the method of multiple isomorphous replacement to solve the phase problem and determine the three-dimensional structure. We have also prepared crystals of the precursor, 7S NGF, which is a complex of three different subunits of composition alpha 2 beta 2 gamma 2. We have collected X-ray data to 3A resolution on two crystal forms with related cell dimensions and orthorhombic spacegroups. Detailed analyses of the structures of NGF in these crystal forms, taken together with data on sequence and biological activity, should give clues concerning the role of the precursor complex in storage and assist the identification of the surface region involved in receptor binding.

Amino Acid Sequence↗
PubMed1989

Structure-function relationships of growth factors and their receptors.

The primary amino acid sequences of several receptor tyrosine kinases have recently made it possible to deduce similarities in the molecular organization of these large multidomain proteins. This has allowed a classification of these receptors into three groups (see Waterfield this Issue and for review in Ref.1). Class I includes the EGF receptor and the neu proto-oncogene, Class II includes the insulin and insulin-like growth factor 1 (IGF-1) receptors, and Class III the platelet derived growth factor (PDGF) and the colony stimulating factor 1 (CSF-1) receptors. The conformation of the ligands for the Classes I and II receptors have been defined using X-ray diffraction, 2-D nuclear magnetic resonance (NMR) and knowledge based modelling procedures. It seems that the ligands are more diverse in sequence than the receptor tyrosine kinases so they cannot be classified as rigorously. However, certain features are common to all growth factors (so far defined) which form compact, globular structures and this allows a discussion of possible interactions between the ligand and receptor; but in the absence of a molecular structure for any of the receptors, we can only review biochemical evidence and deductions from predictive and modelling studies. Various models for the signal transduction process are discussed in the light of current work on receptor interactions.

Growth Substances↗