©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Determinants of Neurotrophin Action (*)

Neil Q. McDonald (1) Moses V. Chao (2)

From the  (1)ICRF Unit for Structural Molecular Biology, Department of Crystallography, Birkbeck College, London WC1E 7HX, United Kingdom and the (2)Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021

INTRODUCTION
Neurotrophin Structure and the Cystine Knot Superfamily
Each Neurotrophin-Receptor Interaction Is Distinctive
Interactions between trk and p75 Receptors
Consequences of Neurotrophin Ablation
Summary
FOOTNOTES
REFERENCES


INTRODUCTION

The neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous systems(1) . Five neurotrophins, NGF, (^1)BDNF, NT-3, NT-4/NT-5, and NT-6, associate as non-covalent homodimers in their biologically active form (2, 3) . These target-derived trophic factors are active on distinct sets of embryonic neurons, whose dependence is in some cases restricted in duration(4) . The neurotrophins are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury, either individually or in combination with other trophic factors such as ciliary neurotrophic factor or fibroblast growth factor (5, 6) .

Responsiveness of neurons to a given neurotrophin is governed by the expression of two classes of cell surface receptors(7) . For NGF, these are p75 (p75) and p140 (referred to as trk or trkA) receptor tyrosine kinase. The related trkB receptor tyrosine kinase binds both BDNF and NT-4/5(8) , and the trkC receptor binds only NT-3(9) , while p75 is able to interact with each neurotrophin with equal affinity, though with different rate constants(10) . After binding ligand, the neurotrophin-receptor complex is internalized and retrogradely transported in the axon to the soma(11) . Both receptors undergo ligand-induced dimerization (12, 13) that activates multiple signal transduction pathways. These include the ras-dependent pathway utilized by trk(14, 15) to mediate neurotrophin effects such as survival and differentiation(16, 17) , while p75 activates less well understood pathways (18, 19) and can modulate trk activity(20, 21, 22) .

We focus here on recent progress in defining 1) neurotrophin protein structure; 2) the molecular determinants of neurotrophins important for receptor interaction; and 3) functional properties of neurotrophins determined from gene targeting experiments. For more comprehensive and detailed reviews of these and other aspects of neurotrophin function the reader is referred elsewhere (7, 23, 24, 25) .


Neurotrophin Structure and the Cystine Knot Superfamily

The first view of a neurotrophin molecule has been provided by the structure of NGF. The dimeric NGF possesses a novel tertiary fold that results in a rather asymmetric molecule with dimensions of 60 25 30 Å (26) (Fig. 1A). Each NGF subunit is dominated by two pairs of anti-parallel beta-strands that contribute to the molecule's flat, elongated shape (Fig. 1B, beta(1)-beta(4)). These beta-strands are connected at one end of the protomer by three short loops. These loops are known to be highly flexible from the variability of their conformations observed within independently determined structures of NGF in different crystalline environments(26, 27) .


Figure 1: Three-dimensional representation of NGF. A, the NGF dimer with each subunit colored differently for clarity. B, the tertiary fold for the NGF subunit. beta-Strands are labeled according to (26) . The three disulfide bridges are drawn as whitesticks with green indicating the sulfur- atom. C, electrostatic potential at the surface of NGF and various modeled neurotrophins (adapted from (74) ). Blue indicates positive potential and red indicates negative. This figure was made using GRASP(75) .



Toward the opposite end of the protomer, the three disulfide bridges are clustered and may provide rigidity to this portion of the structure. The topological arrangement of the disulfides is quite unusual (Fig. 1B). Two of the disulfide bridges and their connecting residues form a ring structure (Cys-Cys, Cys-Cys) through which the third disulfide bridge (Cys-Cys) passes to form a tightly packed ``cystine knot'' motif(28) . Fig. 1A shows the parallel orientation of the NGF subunits. This arrangement allows the two pairs of beta-strands from each protomer to pack against each other, generating an extensive subunit interface. The interface has a largely hydrophobic character comprised primarily of aromatic residues, consistent with the tight association constant (10M) measured for NGF(29) .

Each neurotrophin gene encodes a mature sequence of approximately 120 amino acids, rich in basic residues(3, 30, 31, 32, 33, 34, 35) . Of the 24 neurotrophin sequences currently available, only 28 residues are invariant(24) . From the structure of NGF it is evident that the majority of these residues play key structural roles either within the subunit core or the dimer interface, while the remainder display positive angles or are involved in hydrogen bonding or salt bridge interactions. The preservation of these features indicates that the neurotrophins will adopt similar conformations to that of NGF, as has been recently demonstrated(36, 37) . Further indication of the conserved neurotrophin structure, in particular the dimer interface, is seen by their ability to form mixed heterodimers with other neurotrophins(38, 39, 40) . It is not certain at this time whether these heterodimers form and are fully active in vivo.

Quite unexpectedly, the NGF tertiary fold and ``cystine knot'' motif have been identified in structures of transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF), and more recently in human chorionic gonadotrophin (hCG)(41, 42, 43, 44, 45) . Members of this diverse structural superfamily of ligands typically form homo- or heterodimeric species. However, each representative of known structure shown in Table 1has a different subunit arrangement and intersubunit bonding pattern within its respective dimer(46) . This diversity is also seen functionally since each ligand activates quite different receptor classes and signaling pathways. Other putative members of the cystine knot superfamily include the Norrie disease protein, mucin, and von Willebrand factor(47) .




Each Neurotrophin-Receptor Interaction Is Distinctive

As the earliest event in signal transduction by the neurotrophins is the interaction with p75 or trk receptors, these molecular recognition events have been the focus of intensive investigation. The availability of purified ligand and receptor components and cell lines expressing individual receptor types has allowed for affinity cross-linking, equilibrium binding measurements, and site-specific mutagenesis of neurotrophin sequence.

Three vertebrate trk receptor genes have been isolated, including numerous variants of trk structure(48) . Each neurotrophin binds preferentially to a specific trk but also possesses some ability to interact with other trk family members(7) . BDNF and NT-4/5, the most closely related neurotrophins from phylogenetic analysis, are preferred ligands for trkB(49) , but NT-3 and NT-4/5 can also bind to trkA receptors (8, 31) . Two important questions are raised. What are the amino acid requirements for neurotrophin binding to each receptor? Second, how are the apparent trk specificities encoded within the structure of each neurotrophin?

To address these questions, chemical and genetic modification approaches have been used to probe specific amino acid residues, either through the replacement of contiguous stretches of amino acids or by altering groups of residues that are chemically or spatially related (24) . The biochemical and biological consequences of these changes to NGF have been assessed by measuring receptor binding affinities, trk autophosphorylation, neurite outgrowth, and neuronal survival. While some losses in bioactivity have been detected, other mutations have led to new specificities(16, 50, 51) . These approaches have led to an emerging picture of key receptor binding determinants on the NGF molecule.

Mutation of three lysine side chains (Lys, Lys, and Lys) to alanine within NGF resulted in a complete loss of binding to p75 but maintained binding to trkA and produced a survival response(50) . These data confirmed earlier predictions of the role of electrostatic interactions between NGF and p75, made based on their opposite overall electrostatic charge and the clustering of positive charge on the surface of the NGF(26) .

It is noteworthy that these three lysine residues in NGF are not conserved among all neurotrophins. However, modeling of each neurotrophin suggests the preservation of a positively charged surface in a topologically similar region throughout the family (52) (Fig. 1A). Substitution of residues contributing to each of these positive patches to alanine has indicated a role in binding each neurotrophin to p75(51, 52) . Small local structural differences could account for the distinct association and dissociation rates exhibited by NGF, BDNF, and NT-3 for p75(10) . Alternatively, it is possible that additional neurotrophin-specific residues may also make contributions (51) .

Intriguingly, very few single point mutations to alanine within NGF perturb binding to trk A, suggesting that this interaction involves a large number of contacts(16, 24, 51) . Analysis of N-terminal truncations of NGF(53) , site-specific NGF mutants(54) , and novel chimeric neurotrophins (16, 55) has established the importance of the N terminus of NGF in binding to trkA. Also, the C-terminal residues 112-118 of NGF may play a less specific role in binding trkA(16, 56) . In contrast, analogous experiments using chimeric BDNF and NT-3 neurotrophins demonstrate that their N termini are not essential for binding to their respective trks(16, 51) .

Other contacts between NGF and trkA are present in the beta-hairpin loops, including residues 31 and 40-49 (in particular Glu and Asn) and residues 91-97(16, 57) . The intrinsic flexibility of these loops and the N and C termini of NGF and their apparent importance for trkA interaction(26, 27) suggest conformational changes may occur on formation of a productive NGF-trk complex. Similarly positioned residues within these hairpin loops for BDNF contribute to trkB interaction(16, 57) . Less flexible regions of structure are also important, in particular several surface accessible residues within beta-strands 3 and 4 of the main framework of NGF(16) . A similar conclusion was reached for NT-3, suggesting that this surface may be used by all neurotrophins to engage their respective trk receptors(s)(16, 51) . It is clear from these data that despite some notable differences in the manner in which various neurotrophin-trk receptor pairs interact, in each case the binding determinants extend over a large surface area that takes advantage of the neurotrophin's elongated shape(16) . There is however, a conspicuous absence of a conserved set of residues on the surface of each neurotrophin that could define a common functional trk receptor interaction site. This is curious in light of the extraordinarily high conservation found for species variants of certain neurotrophins, such as NT-3. One rationalization is the parallel evolution of distinctive neurotrophin-trk specificities. Structurally, the ideal candidates for determining specificity are the variable hairpin loops and the N and C termini. These regions are solvent-accessible and can accommodate sequence variation without perturbing the three-dimensional structure. They would therefore be capable of adapting functionally to interact with a specific trk and could allow for optimization of individual neurotrophin-trk contacts, thus explaining the apparent differences between trk receptor interactions for different neurotrophins(16, 51, 57) . Insight into neurotrophin specificity for the trk receptors will come from structural studies of complexes of neurotrophins bound to their preferred trk ectodomains.

Based upon the common structural features of neurotrophins, the trk receptor kinase family, and the interrelatedness of p75 and the p55 TNF receptor(58) , speculative models for p75 and trkA receptors can be proposed (Fig. 2) from the known three-dimensional structures of related modular sequences. It is generally assumed that the intrinsic symmetry of NGF is used to cluster individual receptors leading to receptor dimerization and, in the case of trk, subsequent activation via an autophosphorylation mechanism(13, 15) . Dimerization of trk may require the extracellular immunoglobulin-like domains, which are also necessary for NGF binding(59, 60) . From the known biochemical and binding properties, it is plausible that homo- and heterodimeric complexes may be formed by neurotrophin factors or by either receptor type.


Figure 2: Hypothetical model for neurotrophin receptor structures, illustrating both the relative size of the ligand and receptor components using ``borrowed'' structures for the various receptor domains(75) . Both receptors are shown as monomers in their ligand-free state but form specific homodimers in the presence of ligand(13, 15) . The p75 model was built based on the structure of the TNF p55 receptor (58) with the four repeated domains highlighted. Sequence analysis of the trk ectodomain has revealed a modular mosaic extracellular portion(76) . Known structures of relatives of these modules were used to construct a model of trkA, including 1) the leucine-rich repeat, LLR(79) ; 2) two consecutive C2 IgG domains; and 3) the intracellular domain of the insulin receptor tyrosine kinase domain (78) . This figure was generated using SETOR(79) .




Interactions between trk and p75 Receptors

Receptor-mediated signal transduction for the neurotrophins is unique among polypeptide growth factors since two different trans-membrane proteins exist for each neurotrophin. In addition to trk family members, the p75 receptor is a member of the family of receptors represented by TNF, CD40, Fas, and CD40.

From Scatchard analyses, two classes of NGF binding sites exist on the surface of responsive neurons(61) . These sites differ 100-fold in equilibrium binding constants, which can be further distinguished by the rates of ligand dissociation(62) . The proteins responsible for the high affinity NGF binding site have been the subject of considerable debate, since p75 and trkA each exhibit predominately low affinity binding and a small percentage of high affinity sites has been detected for trkA(13) . The contribution of each receptor type to the high affinity NGF binding site has been clarified by kinetic analysis(63) . Whereas p75 displays fast rates of association and dissociation with NGF, trkA interacts with much slower on and off rates. Due to its unusually slow on rate, NGF binding to trkA results in a K of 10-10M. A similar K has also been determined for BDNF binding to trkB(64) . These affinities are distinct from the high affinity binding site, K = 10M, measured in sensory neurons(61, 64) .

When trkA and p75 receptors are co-expressed, the on rate is accelerated 25-fold, creating a new kinetic site whose features are consistent with the high affinity NGF binding site (K = 10M). This site requires an excess ratio of p75 to trk(65) . Hence, one function of p75 is to increase the binding affinity of NGF. It should be emphasized that the interactions of BDNF, NT-3, and NT-4/5 with cognate trk receptors or with p75 may be different from those exhibited by NGF, as noted by mutagenesis experiments.

In addition to binding, signal transduction by trkA can be influenced by p75(20, 21, 22, 66) . Cell culture experiments indicate that p75 is capable of enhancing trkA autophosphorylation(21, 22) . A potential function of the p75 receptor may be to increase the effective concentration of neurotrophin at the cell surface in order to enhance trk binding(21) . Another model is that an altered conformation of trk may be formed in the presence of p75, which facilitates ligand binding and subsequent signaling functions (65) .

Direct interactions between p75 and trk receptors have been difficult to document biochemically. However, immunoprecipitation experiments carried out in cross-linked spinal cord and brain tissues with I-NGF suggest that an association between the trkA and p75 may take place(67) . Photobleaching experiments following a fluorescently tagged p75 receptor have also revealed a potential physical interaction with trkA(68) .

NGF binding to p75 can cause the hydrolysis of sphingomyelin to ceramide(18) , raising the possibility that signaling through p75 could occur independently of trk. Changes in lipid composition may be generated through p75 to create a different membrane environment for trk receptors. Finally, apoptosis may be promoted by action of a ligand-independent form of p75 (19) during specific developmental and physiological conditions. This activity is supported by similarities in the C-terminal domains of p75 with the Fas antigen and the p55 TNF receptor(69) , which are both capable of mediating cell death.


Consequences of Neurotrophin Ablation

A powerful genetic approach to the study of ligand-receptor interactions has been to analyze mouse mutations created by homologous recombination. Although germ line mutation of many vertebrate genetic loci does not necessarily lead to a detectable phenotype, inactivation of the three mammalian trk genes and their ligands results in considerable neuronal cell loss in mutant mice (Table 2). These significant cell losses provide a striking verification of the biological effects of each member of the NT family and the binding specificities of each neurotrophin.



Several general conclusions can be made regarding null mice in which the expression of neurotrophins or their receptors has been eliminated. First, there is a striking agreement in the phenotypes displayed by NGF, BDNF, and NT-3 mutant mice and homozygous mutations in trkA, trkB, and trkC (Table 2). The defects in each case correlate with the cellular populations known to express each neurotrophin and its cognate receptors(25, 48) . Second, targeted disruptions of the trk receptor genes lead to a slightly more severe phenotype than ligand mutations, measured by the extent of cell death and the target populations that are affected. This difference in phenotype suggests that individual neurotrophin ligands may be more dispensable than individual receptors and emphasizes the ability of multiple neurotrophins to interact with a single receptor. Finally, survival of cells in the central nervous system (CNS) in null mutant mice appears to be relatively unaffected compared with the peripheral nervous system (PNS).

Despite overall structural similarities, neurotrophins do not display the same effects with each trk receptor or with the p75 receptor. NT-3, for example, can bind to all three trk receptors, but gives different trophic responses(8, 70) . This behavior may reflect the ability of neurotrophins to bind multiple trk receptors by using different receptor binding sites. The divergent dependency of p75-deficient neurons toward NGF, BDNF, and NT-3 in culture (71) may be attributed to the subtle differences in the binding properties of each neurotrophin for p75, largely governed by their electrostatic surfaces (Fig. 1C). The rates of dissociation are known to be markedly different, with NGF > NT-3 > BDNF(10) . These differences may also account for the differential transport of NGF, BDNF, and NT-4/5 in sensory neurons(72) . Another important criteria is the ratio of p75 to trk family members, which may dictate the degree of responsiveness to individual neurotrophins(22, 65, 70) . It is conceivable that the binding properties exhibited for p75 and trk receptors may result in distinctive consequences in downstream biological responses.


Summary

Five decades of research on NGF have led to the discovery of a small family of evolutionarily conserved proteins, which have vital functions in the survival and neuronal development of specific neuronal populations. The generation of mice lacking neurotrophin expression has recapitulated classic experiments using anti-NGF antibodies to dissect the physiological effects of trophic factor deprivation(73) . Very similar outcomes resulted from both the NGF immunodepletion experiments and the transgenic mouse experiments. The genetic results also verify the structural predictions made from binding results in heterologous cells.

The findings in cell culture and animal experiments clearly indicate the efficacy of neurotrophic factors for promoting the survival of prominent neuronal populations such as sensory and motor neurons. The high degree of conservation of neurotrophin structure is accompanied by a surprising variation in the amino acid contacts used by each neurotrophin with p75 and the trk receptor family members. It is this variation that may provide specificity for each ligand-receptor complex. The future challenge will be to make use of this knowledge to design effective therapeutic strategies to treat neurodegeneration and nerve injury.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995.

(^1)
The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT-3, NT-4/NT-5, and NT-6, neurotrophin-3, -4/-5, and -6; TNF, tumor necrosis factor; NTR, neurotrophin receptor; TGF-beta, transforming growth factor beta; PDGF, platelet-derived growth factor; hCG, human chorionic gonadotrophin; trk, tropomyosin receptor kinase; CNS, central nervous system; PNS, peripheral nervous system.


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