Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Timm, David E.; Ross, Alonzo H.; Neet, Kenneth E.

doi:10.1002/pro.5560030310

Cited by 16 publications

(8 citation statements)

References 66 publications

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“…We find that CH12 has a 15-fold decreased ability to compete with wt NGF on p75-expressing A875 cells, indicating that the NGF COOH terminus contains structural features that cannot be substituted by the homologous BDNF sequences. This supports the recent suggestion that p75 recognizes the different neurotrophins as related but not identical ligands (32,33).…”

Section: Figsupporting

confidence: 91%

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The Role of the Nerve Growth Factor Carboxyl Terminus in Receptor Binding and Conformational Stability

Krüttgen¹,

Heymach

Kahle³

et al. 1997

Journal of Biological Chemistry

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The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116 -120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu 112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH 2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala 116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116 -120, are important for both the structural stability and biological activity of NGF.The neurotrophins are a family of survival and differentiation factors that exert a profound influence on development and maintenance of the nervous system (1). This feature makes neurotrophins promising candidates as therapeutic agents for the treatment of neurological disorders (2, 3). The crystal structure of the prototype neurotrophin, NGF, 1 revealed that it belongs to the cystine knot superfamily of structurally related growth factors (4). The well conserved "core" of the NGF molecule consists of two pairs of anti-parallel ␤-strands that form an elongated dimer interface and are held together by three disulfide bridges uniquely arranged in a characteristic cystine knot motif. The ␤-strands are connected to the "north" of the protomer by three ␤-hairpin loops, whereas the NH 2 and COOH termini appear to be located closer to the "south" of the protomers (see Fig. 7 for details). The loops and termini were not well resolved by x-ray crystallography and are thus likely to be highly flexible (5, 6). Moreover, these domains are regions of highest sequence divergence among neurotrophins. There is ample evidence for an involvement of these neurotrophin regions in specific receptor binding (7).Two NGF receptors have been identified: the p75 neurotrophin receptor and the tyrosine kinase TrkA (8 -10). Although TrkA is able to elicit NGF responses by itself in some systems, p75 can co-operate with TrkA to increase NGF binding to and activation of TrkA at low ligand concentration (11,12). Mutagenesis studies have previously demonstrated that the binding of NGF to p75 is ...

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Section: Figsupporting

confidence: 91%

“…Previous studies highlighted the importance of basic residues in neurotrophins for binding to p75 (Lys 32 (27,28,30). Because Leu 112 is surrounded mostly by uncharged amino acid residues, nonionic interactions are also likely to play a role in the binding of NGF to p75.…”

Section: Figmentioning

confidence: 99%

The Role of the Nerve Growth Factor Carboxyl Terminus in Receptor Binding and Conformational Stability

Krüttgen¹,

Heymach

Kahle³

et al. 1997

Journal of Biological Chemistry

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“…Preliminary investigation of changes in model structure upon complex formation suggests that small changes may occur in loop side chains and at intersubdomain hinges. The magnitude of these changes appears consistent with CD data (Timm et al, 1994). Signal propagation through the structure can also be explored, testing the hypothesis that conformational changes cascade through stacked loops.…”

Section: Applications Of the Modelsupporting

confidence: 65%

Modeled structure of the 75-kDa neurotrophin receptor

Chapman

Kuntz

1995

Protein Sci.

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Motifs in ligand-binding domains of the neurotrophin (NTR) and lymphotoxin (TNFR-I) receptors define a family of receptors that mediates programmed cell death. We have explored relationships of architecture and function in this family through a molecular model of NTR, also called p75NGFR or LANR. Modeling by homology took advantage of four modular subdomains in the crystal structure of TNFR-I that also occur in NTR. Hypothetical complexes between the model and a ligand structure (for nerve growth factor, NGF) were then examined using docking software. NTR appears to bind in the dimer interface of NGF, making two sets of contacts. NTR subdomains 111 and IV provide the ligand-contact surfaces, in contrast to TNFR, in which subdomains 11 and III contact TNF-6. NTR subdomain I1 appears to have been evolutionarily modified, potentially contributing to an interface between receptor subunits. These and other specific predictions of the model will require experimental confirmation.

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“…Data were collected and quantitated with a Molecular Image System (BioRad) to give an apparent EC50 of 184 nM. This apparent value is high because of the experimental conditions of a very high TrkA-RED concentration of 180 nM that is necessary for adequate sensitivity of the crosslinked band (Timm et al, 1994). After appropriate corrections for a stoichiometric titration of a tightbinding competitor (Williams & Momson, 1979), the data are consistent with a Kd in the range of 1 to 10 nM, which is in accord with our other data and with previous results observed with intact TrkA ectopically expressed on Sf21 insect cells (Woo et al, 1995).…”

Section: Formation Of Ngf-trka-red Complexmentioning

confidence: 99%

“…We have previously shown a small change in conformation by CD measurements when each of the neurotrophins interacts with the common low-affinity receptor, p75LANR (Timm et at., 1992(Timm et at., , 1994. A similar experiment was conducted to determine if a conformational change occurred when TrkA-RED bound NGF.…”

Section: Circular Dichroism (Cd) Analysis Of the Ngf-trka-red Interacmentioning

confidence: 99%

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)

1998

Self Cite

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Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and pl4OTrkA. NGF binding to pl4OTrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a PI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 X lo5 M" s -' and 1.6 X s", respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.Keywords: mutant; nerve growth factor; neurotrophin; receptor binding; receptor extracellular domain; recombinant; structure The growth factor (NGF) and its homologous neurotrophic factors are members of the growing family of neurotrophins that play a profound pharmacological role in development and survival of neuronal cells (Snider, 1994). Five members of the neurotrophin family have thus far been isolated, with NGF as the prototype for this family. The other members include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 ~ istry, Finch UHS/Chicago Medical School, 3333 Green Bay Rd., North Reprint requests to: Kenneth E. Neet, Department of Biological ChemChicago, Illinois 60064; e-mail: neetk@mis.finchcms.edu.Abbreviations; BDNF, brain-derived neurotrophic factor; CM5, carboxymethylated dextran matrix; DSS, disuccinimidyl suberate; EDC, N-ethyl-N'-(dimethylaminopropyI)carbodiimide; HBS, HEPES buffered saline; LANR, low-affinity neurotrophin receptor; NGF, nerve growth factor p subunit; NT3, ueurotrophin-3; NHS, N-hydroxysuccinimide; NT4/5, neurotrophin-4/5; PC12, pheochromocytoma 12 cell line; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sf21, Spodoptera frugiperda 21 (insect cell line); TrkA-RED, TrkA receptor extracellular domain (NT4/5); fish neurotrophin-6 (NT6) is the most newly discovered neurotrophin (Ip...

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Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Cited by 16 publications

References 66 publications

The Role of the Nerve Growth Factor Carboxyl Terminus in Receptor Binding and Conformational Stability

The Role of the Nerve Growth Factor Carboxyl Terminus in Receptor Binding and Conformational Stability

Modeled structure of the 75-kDa neurotrophin receptor

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)

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