Notch signaling controls diverse developmental decisions of central importance to cell activity. One of the conserved positive regulators of Notch signaling is Neuralized, the E3 Ubiquitin ligase enzyme that regulates signaling activity by endocytosis. Neuralized has two novel repeats, NHR1 and NHR2, with a RING finger motif at the C-terminus. Both endocytosis of the Notch ligand, Delta, and inhibition of Notch signaling by Tom, a bearded family member, require the NHR1 domain. Here we describe the first crystal structure of NHR1 domain from Drosophila melanogaster, solved to 2.1 A resolution by X-ray analysis. Using NMR and other biophysical techniques we define a minimal binding region of Tom, consisting of 12 residues, which interacts with NHR1 and show by interfacial analysis of protein monolayers that NHR1 binds PI4P. Taken together, the studies provide insight into molecular interactions that are important for Notch signaling.
The neurogenic genes of Drosophila melanogaster encode elements responsible for the cell-cell communication process that determines choice between epidermal and neuronal cell fates. One such gene, neuralized (neur), encodes a 753 amino acid peripheral membrane protein consisting of two Neur homology repeats (NHR1 and NHR2) and a C-terminal RING finger motif. Genetic analysis suggests that this gene is required for the activity of the Notch ligand Delta for Neuralized-mediated endocytosis and Notch-dependent signaling [1,2]. The activity of Delta depends on the endocytic events for normal trafficking which, in the absence of Neuralized, are greatly compromised by accumulation of inactive protein at the cell surface [1,3]. Drosophila Neuralized exists as a complex with Delta, which it ubiquitinates in a RING-finger-dependent manner to promote Delta endocytosis and signaling. Over-expression or suppression of Neuralized has been shown to result in increased or decreased Delta endocytosis and Notch-dependent signaling, respectively, suggesting the obligatory role of Neuralized in Notch signaling [4,5].
Recently, discovered members of the Bearded (Brd) family of proteins have been shown to directly regulate the Notch signaling pathway. These proteins are expressed in areas of active Notch signaling and have at least one high affinity binding site for Notch activated Suppressor of Hairless protein [
In addition to interactions with Delta and Bearded proteins, Neuralized also interacts with phosphoinositides, phosphorylated derivatives of phosphatidylinositol (PtdIns), which are phospholipids present in minor amounts in the eukaryotic cell membrane. This interacttion localises the Neuralized peripheral membrane protein at the plasma membrane in the absence of Delta [9,10]. Phosphoinositides are involved in classical signal transduction at the cell surface, including regulation of membrane trafficking, mitogenesis and apoptosis as well as endocytosis. Phosphatidyl inositol phosphates (PIP) exist in multiple forms, phosphorylated at positions 3 - 5 of the inositol ring. The interaction of phosphoinositides has been localised to a lysine-rich region in the N-terminus of Neuralized, while the NHR1 domain is suggested to be dispensable for the interaction [
Here we describe the crystal structure of NHR1 at 2.1 Å resolution, define the stoichiometry and affinity of the interaction of NHR1 with Tom and show that NHR1 binds to phosphatidyl inositol-4-phosphate (PI4P) in solution and when adsorbed below lipid monolayers. Taken together, the studies provide insight into the structural features of NHR1 in the crystal state and the interactions mediated by this domain that are important for Notch signaling.
PCR-amplified DNA coding sequence of Drosophila melanogaster Neuralized gene, residues (106 - 260) were ligated into the BamH1 and Xho1 sites of pRSFDuet1 vector (Novagen). Protein expression was carried in BL21 (DE3) pLysS cells at 15˚C in LB. The protein expressed as a N-terminal 6XHis-tag was purified from the bacterial lysate using affinity, and size exclusion chromatography. Protein containing fractions were analysed on SDS PAGE, and then concentrated in buffer containing 50 mM Tris/HCl, pH 8.0, 300 mM NaCl and 1 mM DTT.
A protein calibration curve was prepared using protein standards of low molecular weight (calibration kit, GE Healthcare) to estimate the molecular mass of NHR1. These included Apotropin (6.5 kDa), Ribonuclease A (13.7 kDa), Carbonic Anhydrase (29 kDa), Ovalbumin (43 kDa) and Conalbumin (75 kDa). 1 mg/ml concentration of each standard was prepared in 50 mM Tris/HCl, 300 mM NaCl and 1 mM DTT and analysed on Superdex75 16/60 column (GE Healthcare). The NHR1 containing peaks were also analysed by Mass spectroscopy.
Crystallization was performed using the hanging-drop vapour diffusion method at 20˚C with protein concentrated to 5 - 10 mg/ml. Needle shaped crystals of NHR1 were grown in 0.4 M magnesium nitrate and 10% PEG3350 in 4 days. NHR1 crystals were placed in 30% (v/v) ethylene glycol solution with 20% PEG 3350 and 0.4 M magnesium nitrate prior to flash freezing in liquid nitrogen. Diffraction data was collected at 100 K at ESRF (European Synchrotron Radiation Facility) beamline ID23-1. 180 diffraction images, with a 1˚ oscillation per frame, were recorded, leading to a complete data set with some redundancy. The crystal to detector distance was 296.34 mm and wavelength of 0.95370 Å.
The images were auto-indexed, then scaled and integrated using the programs DENZO and SCALEPACK [
Protein Data Bank accession code: The refined coordinates of the model and the structure factors have been deposited with the Protein Data Bank under the accession code 4KG0. The solution structure [PDB code: 2YUE] was compared with the crystal structure of NHR1 to search for structural differences and similarities between the two. A comparative model of NHR2 was cre-
ated using Modeller [
Fluorescence-based thermal shift assay: A fluorescence-based thermal shift assay [
Isothermal titration calorimetry: A MicroCal VP-ITC titration microcalorimeter was used to perform isothermal titration calorimetry (ITC) in order to determine binding parameters between NHR1 and Tom. 1.4 ml of 4.5 μM Tom peptide was placed in the cell and 500 μl of 670 μM NHR1 protein was filled in the titrating syringe. Both the samples were prepared in 50 mM Tris/HCl, pH 8.0, 300 mM NaCl and 1 mM DTT and the experiment performed at 25˚C. A total of 25 injections of 10 μl each were applied into the cell, except that the first 5 μl was discarded. The stirring speed during the titration was 300 rpm. Titrations of peptide to buffer were performed to allow base-line corrections. The Origin software was used to analyse the binding kinetics. The best-fit values of the heat of binding, the stoichiometry of binding and the dissociation constant were determined from the plots of heat evolved per mol of NHR1 injected versus the NHR1-Tom peptide molar ratio using this software.
Lipid binding strips: 10 μg/ml of the purified NHR1 protein was incubated with the membrane lipid strips (Echelon Biosciences) at room temperature. Following the manufacture’s protocol, the strip was first incubated for 1 hour at room temperature with anti-His antibody (GE Healthcare), washed and then incubated with secondary anti-mouse alkaline phosphatase conjugate antibody (Promega) for 1 hour at room temperature. Prior to protein detection, the strip was washed as in the previous step to remove excess antibody and then detected by BCIP/NBT (5-Bromo 4-Chloro 3-Indolyl Phosphate/ Nitro Blue Tetrazolium) alkaline phosphatase tablet (Sigma) after 5 minutes of incubation at room temperature.
A circular trough (S = 20 cm2) was used to prepare the monolayers and the surface pressure was precisely measured with a sensor using a Wilhelmy plate. Null ellipsometry was used to determine the excess concentration of adsorbed molecules by measuring the changes in the polarisation of light upon reflection. A house-made ellipsometer with a He-Ne laser (λ = 632.8 nm) and a polariser were used to record the ellipsometric measurements. The angle of incidence of light on the surface of solution was 1˚ away from the Brewster angle. After reflection on the water surface, the laser light was passed through a λ/4 retardation plate, analyser and a photomultiplier. In this “null ellipsometer” configuration [
The lipids are spread at the surface of the trough and the quantity of spread molecules adjusted to record the starting surface pressure. In the second step, the protein is injected in the subphase (with a needle); the adsorption kinetic is followed by recording the ellipsometric angle and the surface pressure over time. The increase of ellipsometric angle from the initial value of lipid alone to the final value corresponding to the end of the adsorption kinetics of the protein indicates the presence of protein below or inside the lipidic layer. The shear elastic constant was determined by surface rheology. The rigidity of the interfacial layers at the air/water or lipid/water interface was measured using a 10 mm diameter paraffin-coated aluminium disk float, placed at the centre of a 40 mm diameter Teflon trough [22-24]. A small magnet in the float was kept centred by permanent magnetic field. A mirror fixed on the magnet reflected the laser beam onto a photodiode that helped in sensitive angular detection of the float. When a sinusoiddal torque of 0.01 - 100 Hz was applied to the float by an oscillating field perpendicular to the permanent solenoid field, the device acted as a simple harmonic oscillator. The latter field acted as a restoring torque equivalent to an interfacial layer with a rigidity of 0.16 mN/m (set as the sensitive limit of the rheometer) and the resistance of the interfacial layers was directly measured. One important advantage of this set-up is the achievement of a high sensitivity due to very small deformations. To assess and detect rigidity of the monolayer, the amplitude and phase of the mechanical response of pure subphase was first analysed in the frequency range of 0.01 - 100 Hz. This measurement took approximately 1 hour. Then, the protein solution was directly poured in the trough and the mechanical response of the layer formed at the interface recorded at a fixed frequency of 5 Hz. At the end of the kinetics, when the shear elastic constant (expressed in mN/m) reached a constant value, a new measurement between 0.01 and 100 Hz was recorded to determine whether the system behaves as an elastic layer. Rigidity measurements were carried out in parallel to ellipsometry. All of the experiments were performed at 18˚C. Protein solutions were prepared in the range 1 - 80 µg/ml (i.e., ≈36 nM – 3 µM) in 50 mM Tris/HCl, pH 8.0, 300 mM NaCl and 1 mM DTT for surface pressure, null ellipsometry and surface rheology measurements.
The Neuralized NHR1 domain of Drosophila melanogaster was crystallised with a tandem of six histidine residues (His-tag) at the N-terminus. The crystal structure of NHR1 was determined to a resolution of 2.1 Å by molecular replacement and refined Rcryst of 16.1% and Rfree of 22.3%. The data processing statistics, the refinement statistics and an assessment of the model geometry are included in
Before we defined the crystal structure, homologues for the NHR1 domain had been identified using the sequence-structure homology recognition program, FUGUE [
Results obtained from DALI search for the NHR1 domain corroborated the FUGUE analysis. Structural superposition and alignment of the SPRY domain proteins with NHR1 using Baton/Comparer showed that the secondary structure elements, mostly β strands, were organised in a similar way. The common residues between both the domains were compared using JOY [
The SPRY domain occurs in a variety of cellular proteins that mediate protein-protein interactions. This domain is considered to be a migratory domain since it is found associated with different protein domains, and recognises a specific individual partner. SPRY is known to interact with a peptide called VASA [
From structural analysis of NHR1 it was evident that the overall domain is similar to SPRY domain. The NHR1 domain mediates nuclear envelope association and delta-dependent inhibition of nuclear import and is involved in interaction with Bearded and lipids [7,9,30, 31]. We used an evolutionary trace analysis method Trace Suite II [
crystal structure of the NHR1 domain. The evolutionary trace analysis shows that conserved residues that might interact with binding partners to carry out the protein’s function clustered in the loop region of NHR1 domain.
The sequence identity between NHR1 and NHR2 is 27%, allowing a comparative model of NHR2 using modeller [
An additional approach to the study of these solvent exposed residues was adopted. This involved examining inter-molecular interactions between symmetry related NHR1 molecules in the crystals using the PICCOLO software [
Interestingly, size exclusion chromatography and mass spectrometry revealed the existence of monomeric (21.25 and 19.39 kDa, respectively) and dimeric (38.763 and 44.7 kDa, respectively) forms of NHR1 (
hybrid system to establish if NHR1 can associate to forms homodimers and whether NHR1 and NHR2 physically interact. In both cases, yeast two-hybrid failed to demonstrate any interaction (data not shown).
Despite numerous attempts, we were unable to obtain a soluble and folded form of Tom protein (Gupta D., PhD Thesis) [
An increased melting temperature of ~2 degree of NHR1 in the presence of the peptide implies that the two molecules interact with each other and form a complex. The control peptide showed no increase in the melting temperature, suggesting that it does not bind the NHR1 protein, confirming that the interaction of NHR1 with Tom peptide is specific. Furthermore, the sigmoidal shape of the melting curve represents a cooperative, twostate transition thermal unfolding of a typical single and autonomously folding protein unit.
Isothermal titration calorimetry (ITC) was performed for determination of the NHR1-Tom binding affinity. Complete saturation of the binding sites with the Tom peptide was achieved when a high NHR1 concentration (670 μM) was used to titrate against 4 μM of the peptide. The binding curve is shown in
The binding interface of NHR1 along with the superimposed NHR2 model and Tom peptide were compared with the binding interface of VASA peptide with SPRY domain protein (PDB code: 2IHS) shown in
The interaction of phosphoinositides (PIP) with Drosophila melanogaster Neuralized protein [
However, since the crystal structure of NHR1 reveals the domain contains a large hydrophobic region, we investigated whether NHR1 is also involved in binding lipids. For this, we performed lipid-binding experiments with purified NHR1 blotted onto nitrocellulose membranes previously spotted with immobilised lipids (membrane lipid strip, Echelon Biosciences). We found that purified NHR1 binds PI4P to a greater extent than phosphatidic acid (
To determine whether NHR1 was surface active, and ascertain its intrinsic amphiphilic nature, experiments were first performed at the air/liquid interface, in which the air is the hydrophobic component of the system. Purified NHR1 was directly dispensed in the trough at a final concentration of 30 μg/ml (1.5 μM).
shows that NHR1 was saturated at the interface when the subphase concentration reached ≥3 µg/ml, strongly suggesting that NHR1 is surface active. Therefore, a bulk concentration lower that 3 µg/ml (i.e. 2.5 µg/ml, 0.13 μM) was chosen as the working concentration for subsequent experiments to determine the interaction of NHR1 with lipid, a value reflecting that protein-lipid interactions are favoured compared to protein-protein interactions. When NHR1 at 2.5 µg/ml in buffer solution is poured in the Langmuir trough, after 8 hours of the experiment the
surface pressure at the interface reached a plateau and remained constant at 13 mN/m respectively. The ellipsometric angle increases with time and reaches a plateau around 9˚, corresponding to a surface concentration of 1.8 mg/m² according to the relationship from De Feitjer: valid in case of globular proteins [
The low protein concentration allows the recording of the first adsorption events and to extract several parameters relevant to the surface activity. Two of these parameters were obtained from the plot of versus p (
Surface rheology experiments were carried out to define the viscoelastic properties of NHR1 monolayers.
To determine whether NHR1 interacts with the lipid PI4P, two monolayers were prepared by spreading solutions of PI4P and DOPC (1,2-dioleoyl-sn-glycero-3- phosphocholine). An initial surface pressure for lipids of p = 25 mN/m was chosen because lipidic layers are rather dense at these conditions and also reduce adsorption of protein in the free space of the surface (this could happen if the initial surface pressure was lower than this value). The results of these experiments are shown in Figures 8(a) and (b).
For DOPC monolayers, the initial surface pressure
corresponded to an ellipsometric angle of, for PI4P the corresponding value was. These values remained stable over a few hours (