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Structure of Nerve Growth Factor Complexed with the Shared Neurotrophin Receptor p75

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Science  07 May 2004:
Vol. 304, Issue 5672, pp. 870-875
DOI: 10.1126/science.1095190

Abstract

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 Å crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.

The neurotrophins (NTs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are a family of secreted growth factors that have a wide range of functions in the development and maintenance of the vertebrate nervous system [reviewed in (1, 2)]. Neurotrophins, which associate into noncovalent homodimers with conserved structural features (1), are capable of promoting neuronal cell survival, or cell death, depending on the context of the neuronal environment (3). Although NTs hold promise as clinical agents, an impediment to improving their therapeutic utility has been an incomplete understanding of the complex molecular mechanisms by which NTs exert their actions (4).

The pleiotropic actions of neurotrophins are mediated by two structurally unrelated classes of receptors, the common neurotrophin receptor p75 and the Trk family receptor tyrosine kinases (1, 5, 6). Each neurotrophin is specific for different Trk receptors, but all bind to p75 with comparable affinities (1). p75 is the founding member of the family of cysteine-rich domain (CRD)–containing receptors exemplified by the tumor necrosis factor (TNF) receptor superfamily (TNFR-SF). However, whereas other TNFR-SF members traditionally bind trimeric ligands like TNF, p75 binds dimeric neurotrophins, which suggests a different recognition mechanism for the ligand and receptor.

p75 can respond to neurotrophins either independently or by modulating the affinity and specificity of the Trk receptors for NTs (1, 7, 8). In the absence of Trk, NT activation of p75 has been shown both to induce neuronal apoptosis (cell death) and to modulate cell survival (6, 8, 9). Both functional outcomes appear mediated by a wide array of different intracellular signaling molecules downstream of p75 [e.g., nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK)], including possible involvement of the p75 intracellular death domain. p75 can also coordinate with Trk receptors to form what have classically been referred to as “high-affinity” NT-binding sites on neuronal cells (1, 10). Experimental evidence exists for both ligand-independent and ligand-dependent interactions between p75 and Trk, as well as allosteric communication between the receptors [reviewed in (8)], to explain the p75-mediated affinity and specificity conversion of Trks (1). However, the molecular mechanisms of the p75-Trk cross talk are not understood (7). The crystal structure of NGF bound to a single ligand-binding immunoglobulin-domain of TrkA revealed a symmetric 2:2 stoichiometry of the complex, establishing neurotrophin-induced dimerization as the Trk activation mechanism (11). There is, however, no structural evidence for a similar clustering mechanism to explain p75 activation.

We describe the crystal structure of NGF in complex with the extracellular domain of p75. The structure reveals a 2:1 NGF/p75 complex in which the second potential p75-binding site is disabled by p75-induced conformational changes in the NGF molecule. This structure also explains the basis of the observed p75 cross-reactivity with all NTs. The formation of an asymmetric complex for all p75-NT interactions, and the apparent ligand-induced disassembly of a p75 dimer, can now help clarify some of the complex functional literature bearing on the molecular mechanisms by which p75 is activated.

Crystals of the complex between NGF and p75 were prepared with a soluble, baculovirus-expressed rat p75 ectodomain (residues 1 to 161) and recombinant human NGF expressed from Escherichia coli. The structure was determined to a resolution of 2.4 Å by combining MIRAS (multiple isomorphous replacement with anomalous scattering) with MR (molecular replacement) phases from a previous model of NGF (see table S1) (12). The asymmetric unit of the crystal contains one NGF homodimer and a single copy of p75 (Fig. 1, A and B). This composition was contrary to our expectation that, similar to other single-transmembrane signaling receptors, ligand binding would induce p75 dimerization (5, 13, 14).

Fig. 1.

Structure of NGF complexed with p75. (A) p75 binds along one side of the NGF homodimer. Backbone representation of NGF monomer A is colored green; monomer B, blue; and p75, purple. Disulfide bonds are shown as green sticks. Other figures have the same coloring scheme. (B) p75 binds across the NGF dimerization interface. Front view, with a horizontal rotation of 90° from the orientation of the complex seen in (A) with NGF depicted as surface and p75 as tube. (C) p75 uses the opposite side as TNFR-SF members for ligand binding, but both bind within their ligands' oligomer interfaces. One copy of TNF-R1 from the TNF-β–TNF-R1 complex (16) is superimposed with p75 from the NGF-p75 complex. The two receptors used in superimposition are depicted in main-chain traces, and the other molecules in space-filling format.

The overall structure of the 2:1 NGF/p75 complex can be described as a roughly parallel configuration, in which the long axis of p75 aligns with the seam formed by the edge of the NGF dimerization interface (Fig. 1, A and B). The complex is tethered together through two spatially separated binding epitopes, which we designate “site I” and “site II” (Figs. 1A and 2, A and B). The site I epitope is formed between the wider, or top, end of the NGF dimer and the first and second CRDs of p75. The site II epitope is formed between the tapered end, or bottom, of the NGF dimer and the junction between the CRD3 and CRD4 domains of p75. Because p75 inserts within the dimeric seam, both p75 binding sites on NGF are formed by contributions from both NGF monomers (Fig. 1B). On the opposing face of the NGF dimer, the identical sets of residues are presented on the corresponding epitopes, which we designate “pseudosites I and II,” as they are not used to bind p75 (Fig. 1A). Because unbound NGF (15) is a symmetric homodimer, both sides of the molecule are equally competent to initially engage p75.

Fig. 2.

The interaction between NGF and p75. (A) Close-up of the site I interface (NGF monomer A is green; monomer B, blue; and p75, purple). The backbone is depicted as tubes, and side chains are depicted as sticks. The buried salt bridge between NGF Lys88A and p75 Asp41 in site I is highlighted by thickened side chains. (B) Close-up of the site II interface. (C) Charge complementarity between the acidic p75 and the basic NGF dimer is shown as their respective GRASP surfaces. Acidic is red and basic is blue. (D) A representative electron density map (σA 2FobsFcalc) around the patch 1 of site I, showing the knob-in-hole surface complementarity. (E) NT sequence alignment showing that the p75 contact residues are conserved across all NTs. Monomer A residues are shaded in green and monomer B residues shaded in blue. Site I residues are labeled “1,” and site II residues are labeled “2”.

The extracellular region of p75, made up of all four CRDs, is an elongated and kinked structure (∼115 Å long) (Fig. 1). A comparison of the NGF-p75 complex with TNF-receptor complexes (16) reveals both significant differences and similarities (Fig. 1C). The first difference is that these two receptors use almost 180° opposite faces to interact with their respective ligands. p75 uses the convex face formed by the curvature of CRDs 1 and 2 to form a tangential contact point with a protruding NGF site I, whereas TNF-R uses the concave face of this same curved region to receive the complementary rounded shape of TNF. The second difference is that TNF-R makes only a single point of contact with TNF, whereas p75 has two contact points with NGF. The third difference is that, in the symmetric TNF-R complexes, the trimeric ligand symmetrically trimerizes three TNF receptors, whereas in the asymmetric p75-NGF complex, the dimeric symmetry of the ligand is not used to dimerize p75. Despite these differences, however, there is an interesting convergent feature of both complexes: Both p75 and TNF-Rs bind along the seam of the interfaces between two monomers.

The NGF bound to p75 closely resembles the parallel homodimeric structure of unbound murine NGF (15). Superimposing the common Cα atoms between p75-bound and unbound NGF reveals that the two NGF monomers in the p75-bound complex have greater structural differences with one another [root mean square deviation (rmsd) 1.1 Å] than between NGFs in different structures; these differences reflect the asymmetry induced by p75 binding (discussed below).

Overall, 2300 Å2 of solvent-accessible surface is buried between NGF and p75, which can be subdivided into 1360 Å2 for site I and 940 Å2 for site II. The site I buried surface is skewed toward contributions from monomer A (monomer A, 59%; monomer B, 41%), whereas site II is the reverse (monomer A, 39%; monomer B, 61%) (Fig. 1B). Consistent with our thermodynamic data showing that NGF binding is mainly entropy-driven (Fig. 3B), both site I and site II bury large areas of hydrophobic surface, with salt bridges and hydrogen bonds providing surrounding complementarity. Charge complementarity is an important feature of p75 ligand recognition, as previously shown by mutagenesis (4). The surface of p75 is covered with a high density of negative charge that complements the overall basic charge of NGF (Fig. 2C).

Fig. 3.

Biochemical evidence of a 2:1 NGF/p75 complex in solution. (A) Elution positions of unliganded p75 versus the NGF-p75 complex from a size exclusion column (Superdex-200, Amersham). The column was calibrated with molecular-mass standards as shown on top. SDS-PAGE gels of the peak fractions are shown in boxes (blue and red boxes and fraction hash marks for p75 alone and p75-NGF complex, respectively). (B) Titration of NGF into p75 with the use of ITC. (C) Measurement of molecular mass of the NGF-p75 complex by MALS. Proteins were injected onto a Superdex-200 gel filtration column, and the output was subjected to real-time MALS analysis. Top, unliganded p75; bottom, the NGF-p75 complex in excess of NGF. For the latter, the left peak corresponds to the p75-NGF complex, and the right peak corresponds to excess unliganded NGF.

The site I contact surface between NGF and the CRD1-CRD2 domains of p75 can be divided into two patches with unique characteristics (Fig. 2A). The first patch (patch 1) involving the p75 CRD2 is a hydrophobic knob-in-hole configuration (Fig. 2, A and D). The NGF side chains Phe101A, Ile31A, Trp21B, and Tyr52B form a pocket in the dimerization seam into which p75 CRD2 inserts the side chains Pro70, Met67, and Ala69 (Fig. 2D). The hydrophobic interactions are enhanced by two hydrogen bonds from the hydrophobic side chains (p75 Asn42 Oδ2–NGF Tyr52B OH and p75 Ser68 O–NGF Trp21BNϵ1). The second patch of site I (patch 2) involves the p75 CRD1-CRD2 junction and features a buried salt bridge between p75 Asp41 and the NGF Lys88A, which is on the bottom of a deep hydrophobic pocket formed by NGF residues Trp99A, Phe49B, Phe101A, and Tyr52B.

The smaller site II interface consists of the p75 CRD3-CRD4 junction interacting with the NGF N- and C-terminal regions and two “bottom” loops (Fig. 2B). The shape of site II is characterized by protruding, interdigitating apolar side chains, forming a zipper-like complementarity within the interface. From top to bottom, NGF Phe12B fits in between the p75 Leu106 and Pro135; NGF Trp76A fits in between p75 Pro135 and Leu137-Pro138, and the NGF His75A-Trp76A residue pair contact p75 Pro138. Two salt bridges (NGF Arg114A–p75 Glu119; NGF Arg69B–p75Asp135), one on each side of the row, may assist in steering the hydrophobic zipper contacts into proper alignment during complex formation.

One of the most important aspects of p75 biology is its role as a shared receptor for all neurotrophins (1). The NGF-p75 structure explains the basis for this cross-reactivity. The human neurotrophin sequences have an average sequence identity of 52%. This homology is reflected in the close structural similarity of neurotrophins whose crystal structures have been determined (0.9 to 1.0 rmsd for common Cα values). Mapping the residues at the site I and site II contacts seen in the NGF-p75 complex onto the neurotrophin sequence alignment (Fig. 2E) shows that the p75 contact regions are well conserved in all four neurotrophins. Thus, shared neurotrophin recognition by p75 does not indicate promiscuous binding, but rather conservation of a precise set of ligand contact residues across the NT family. This contrasts with the shared signaling receptor, gp130, which cross-reacts with highly divergent cytokine residues, using entirely different receptor-ligand contacts, through an enthalpy-entropy compensation mechanism (17).

To ensure that the 2:1 NGF/p75 stoichiometry is not an artifact of crystallization, we measured the ligand:receptor stoichiometry and the assembly thermodynamics of p75 complexes in solution with NGF, NT-3, and NT-4/5, using three independent methods: (i) gel filtration chromatography, (ii) multiangle laser light scattering (MALS), and (iii) isothermal titration calorimetry (ITC). Size-exclusion chromatography of p75 alone reveals it to be a mixture of a higher and lower molecular weight species (Fig. 3A), which were determined by MALS to be dimer and monomer, respectively (Fig. 3C). The p75 monomer:dimer ratio displays a concentration-dependent equilibrium, although the dimer persists even at low p75 concentrations. This solution dimer is consistent with functional studies showing that preformed p75 dimers exist on the cell surface, in the absence of neurotrophin (13, 14).

Comparison of the gel filtration elution profiles of unliganded p75 and the NGF-p75 complex (as well as the NT-4/5–p75 and NT-3–p75 complexes) shows that the NGF-p75 complex elutes later and is, therefore, a smaller molecular weight species than the p75 dimer (Fig. 3A). We see no evidence of high-order (e.g., dimeric) forms of the p75-NGF complex, which suggests that NGF antagonizes p75 dimer formation. We then used calorimetry (ITC) to measure the stoichiometry and binding thermodynamics between p75 and NGF (as well as NT-4/5 and NT-3) (Fig. 3B). A measured stoichiometry of 2.3 NGF monomers to one p75, obtained at various temperatures, supports the crystal structure of one NGF dimer bound to a single p75. The affinity of p75 for NGF and NT-4/5 is lower than the KD measured in cell-based assays (18), but this is commonly observed for receptors when their soluble ectodomains are released from the two-dimensional constraints of the cell membrane and incur an entropic penalty in solution. We also measured the molecular mass of the NGF-p75 and NT-4/5–p75 complexes using MALS in the presence of excess NTs (Fig. 3C). The excess unliganded NGF homodimer molecular mass is 27 kD, whereas the NGF-p75 complex molecular mass is 55 kD, equal to an NGF homodimer plus a p75 monomer (31 kD). The composition of these MALS peaks was confirmed by SDS–polyacrylamide electrophoresis (SDS-PAGE). Similar MALS results were also observed for the NT-4/5–p75 complex. Therefore, three lines of evidence, gel filtration, ITC, and MALS, all indicate that NGF homodimers and other NT dimers bind to only one p75 in solution.

To explain why the accessible, symmetry-related binding site on NGF does not bind to a second p75, we rotated the bound NGF dimer around the two-fold homodimeric symmetry axis to superpose sites I and II with pseudosites I and II (Fig. 4A). This superposition revealed that p75 binding to one side of NGF induces a bending of the entire NGF molecule: The central β sheets remain intact, but the head and tail loops are bent away from p75, leaning toward the pseudosites. As measured from a centrally located pivot point in NGF, there is a 10° relative movement of pseudosite I, and a 6° relative movement of pseudosite II (Fig. 4A). The two-point attachment of p75 to NGF is now rationalized, as it facilitates each site's acting as a fulcrum to enable p75-induced bending.

Fig. 4.

The asymmetry of NGF-p75 complex. (A) NGF is bent away from the p75-binding face toward the disabled pseudosites. The bound NGF dimer is rotated 180° (in gray) and overlaid onto the NGF dimer in the NGF-p75 complex (colored). Rotation angles of the two ends are measured by using lines drawn from a Cα atom at each of the tips of the protruding NGF top and bottom loops (Asn46 and Lys74, respectively), to a Cα atom at the center of each NGF tip-waist junction (Phe53 and Val109). The angle formed by the intersection of these lines is the bend angle we refer to. (B). Comparison of pseudosite I (in gray) with site I (colored), highlighting the numerous differences in positions and orientations of side chains, most notably around Phe49. (C) Comparison of pseudosite II (in gray) with site II (colored), showing a rigid translation and rotation of the entire site without significant intrasite structural perturbations. (D) Diagram of cell surface equilibrium between various neurotrophin, p75, and Trk receptor complexes, including potential model of p75–Trk–neurotrophin trimolecular complex. NGF is depicted as tubes, and the receptors are depicted as surfaces. From high to low TrkA/p75 ratio, the complex could choose to be 2:2 NGF/TrkA, 1:2:1 TrkA/NGF/p75, 2:1 NGF/p75, and finally an unliganded p75 dimer.

The allosteric consequences of p75 binding are numerous coupled structural perturbations in the NGF pseudosites, which lie between 15 and 20 Å away from the initial sites of p75 interaction at sites I and II. Thus, a long-range allostery is transduced through the NGF molecule. In pseudosite I, there is a widening between the two NGF monomers that make up the composite receptor binding site. A 3 Å hinge-like movement of the NGF L2 loop in pseudosite I (residues 41 to 50) would result in a clash with an incoming p75. Collectively, side-chain positions of receptor contacts in both pseudosites I and II are altered as the loop main chains bend away from the bound p75 (Fig. 4C). The combination of pseudosite I and II movements introduces a change in the relative orientations between the two pseudosites (i.e., distance, angular relationship). Given the two-point attachment of p75 to NGF, combined with the apparent rigidity of TNF-R family receptors, we suggest that p75 is intolerant to changes in the relative orientations of the two sites. Attempts to manually dock p75 onto both pseudosites simultaneously resulted in numerous steric clashes and implausible NGF-p75 contact distances relative to those formed at the initial sites of engagement.

The asymmetric interaction mode of p75 and NGF can be contrasted with other asymmetric receptor and ligand complexes. The immunoglobulin IgE Fc receptor complex with Fc (19), the NKG2D complex with MIC-A (20), and the complex of CNP with the natriuretic peptide receptor (21) are all asymmetric complexes containing a symmetric homodimer. However, in these cases, the ligand or receptor binds directly on the two-fold symmetry axis; thereby, it sterically imposes the asymmetry through blockade of the binding site. The p75-NGF complex is distinct in that the receptor binds away from the symmetry axis of NGF, which leaves the mirror-image binding site completely accessible for another receptor interaction. Therefore, an allosteric mechanism is required in order to disable the pseudosites. A possible functional rationale for this mechanism is to preserve an open face of NGF that could engage a second, but different, receptor and form a heterotrimeric complex.

The asymmetric NGF-p75 complex structure now helps provide a framework for interpreting the voluminous body of functional data published on NT signaling through p75 and Trk (13, 5, 79). On the basis of our structural and biochemical data, we propose that one mechanism of p75 activation is through NT-induced dissociation of a receptor dimer. This mechanism is consistent with some experimental data (1, 5, 8), such as a cross-linking study showing that apoptotic induction by p75 requires monomerization, whereas enforced dimerization is inhibitory (22). The NT-induced p75 disassociation mechanism does not exclude the possibility that other receptor configurations or oligomers may initiate signaling, dependent on the cellular environment. For instance, p75 alone, p75 as dependence receptor, p75 with Trks, and p75 with Nogo receptor may each have signaling mechanisms distinct from the NT-p75 complex. This could explain the complexity of p75 signaling. For example, in the same type of neuronal cells, NGF binding to p75 inactivates Rho (23), whereas binding of Nogo to the NgR-p75 complex results in Rho activation (24).

The asymmetric NGF-p75 complex lends some mechanistic insight into the role of p75 in coordinating with Trk. The TrkA-NGF complex is known to comprise two NGF monomers and two TrkA molecules in a symmetric 2:2 stoichiometry (11). Although Trk and p75 are mutually exclusive on the same side of NGF because of the clash between the Trk D5 domain and the p75 CRD2-CRD3 junction, NGF is structurally capable of binding TrkA and p75 simultaneously on opposite sides to form a trimolecular complex (Fig. 4D). Of note, the TrkA binding site on NGF, composed of the central β sheet, is unaffected by the conformational changes induced by p75; these changes are localized to the distal loops of NGF (Fig. 4A). The structural possibility of the trimolecular complex supports an equilibrium between p75 dimers, p75-NT complexes, NT-Trk complexes, and trimolecular Trk–NT–p75 signaling complexes (Fig. 4D) that would be dictated by the relative expression levels of each component and by the concentration of NT (7, 8). Further, the p75-induced neurotrophin conformational changes on the side of the pseudosites could be a mechanistic glimpse of the long-suspected allostery by which p75 modulates Trk ligand affinity and specificity for neurotrophins.

Supporting Online Material

www.sciencemag.org/cgi/content/full/304/5672/870/DC1

Materials and Methods

Table S1

References and Notes

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