Characterization of symmetric complexes of nerve growth factor and the ectodomain of the pan-neurotrophin receptor, p75NTR

Abstract

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.

FIGURE 1

A, scintillation proximity assay of purified p75^NTR with mouse 2.5 S NGF. Best fit is K_d = 2.101 ·10⁻⁹ m, with an R² = 0.9885. B, S200 size exclusion chromatography of p75^NTR, NGF, and the p75^NTR·NGF complex. The colored bars mark fractions analyzed on SDS-PAGE. Molecular mass calibration is indicated at the top.

FIGURE 2. Mass spectrum of the intact p75 ^NTR·NGF complex (68.37 kDa)

Inset shows tandem mass spectrum of the p75^NTR·NGF complex for a broad isolation window centered around the 15+ charge state. Ions labeled on the spectrum correspond to charge states of (blue circles) NGF monomer, 13.27 kDa (red rectangles) p75NTR monomer, 20.5–20.9 kDa, p75^NTR·NGF 2:2 complex, 1:2 p75NTR·NGF complex, 47.45 kDa, and 2:1 p75^NTR·NGF complex, 55.10 kDa (all labeled with the appropriate number and type of either blue circles or red rectangles).

FIGURE 3

A, expanded view of the region between 2100 and 3100 m/z from Fig. 2. Modified versions of p75^NTR are indicated as II, III, and IV (at 20.6, 20.7, and 20.9 kDa), and observed modified NGF is indicated with an asterisk. B, mass spectrum of isolated p75^NTR. Modified versions of the protein are marked to correspond with the assignments from Fig. 3A (see text) including an additional glycoform labeled I (20.45 kDa). C, MALDI-TOF data of glycopeptides resulting from a tryptic digest of p75^NTR (see TABLE ONE for details).

FIGURE 4. Analytical ultracentrifugation data of p75^NTR, NGF, and p75^NTR·NGF complex

p75^NTR (A) and NGF (B) sedimentation velocity measurements. Collected data (top), fringes (middle), and continuous c(M) distribution (bottom) (C) p75^NTR·NGF complex sedimentation equilibrium measurement at 0.25 mg/ml (left panel) and 0.5 mg/ml (right panel). Both concentrations were run at two speeds.

FIGURE 5. Comparison of the merged experimental x-ray scattering curve of the p75^NTR·NGF complex with simulated profiles of 1sg1.pdb (2:1 complex) and two conceptual 2:2 complexes: 1sg12.pdb, derived from 1sg1.pdb, and 1bet2.pdb, derived from 1bet.pdb

The inset displays the distance distribution function p(r) from which a maximum particle dimension of 105 Å is deduced.

FIGURE 6. Molecular models of 2:2 p75^NTR·NGF complex

A and B, SAXS-derived Gasbor18 model of p75^NTR·NGF complex (black mesh) from two different views, superimposed with 1sg12.pdb (as α-carbon trace, NGF in blue and p75^NTR in red). The CRDs and the N-glycosylation sites (Asn⁶⁰) of the human protein are indicated. C, surface representation of the conceptual model 1sg12.pdb with NGF in blue and p75^NTR in red. D, p75^NTR·NGF superimposed with 1sg12.pdb (as in C), showing the observed alternative first CRD conformation.