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Copyright © 2008 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 95, Issue 4, 1928-1944, 15 August 2008

doi:10.1529/biophysj.107.126995

Proteins

Biophysical Characterization of the Unstructured Cytoplasmic Domain of the Human Neuronal Adhesion Protein Neuroligin 3

Aviv Paz^*, †, Tzviya Zeev-Ben-Mordehai^*, †, Martin Lundqvist^¶, Eilon Sherman^‡, Efstratios Mylonas^∥, Lev Weiner^§, Gilad Haran^‡, Dmitri I. Svergun^∥, **, Frans A.A. Mulder^¶, Joel L. Sussman^* and Israel Silman^†, ,  

^* Departments of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
^† Neurobiology, Weizmann Institute of Science, Rehovot, Israel
^‡ Chemical Physics, Weizmann Institute of Science, Rehovot, Israel
^§ Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel
^¶ Department of Biophysical Chemistry, Groningen University, Groningen, The Netherlands
^∥ European Molecular Biology Laboratory, Hamburg, Germany
^** Institute of Crystallography, Russian Academy of Sciences, Moscow, Russia

Address reprint requests to Israel Silman, Tel.: 972-8-934-3649.

The neuroligins (NLs) are a set of neuronal adhesion proteins belonging to the family of cholinesterase-like adhesion molecules (CLAMs). All CLAMs contain an extracellular domain that bears high sequence homology to acetylcholinesterase (AChE), and share the same conserved intrachain disulfides. However, they all lack one or more of the members of the catalytic triad of AChE, and are thus believed to be catalytically inactive 1. To date, three transmembrane CLAMs have been identified: the mammalian and invertebrate NLs, and the insect proteins, gliotactin (Gli) and neurotactin (Nrt) 2 (Fig. 1). All three are single-pass transmembrane proteins whose cytoplasmic domains contain ∼120–320 amino-acid residues, and bear no sequence similarity to any known protein, or even to each other.

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Figure 1

Domain architecture of the CLAMs. All CLAMs contain a ChE-domain (shaded), which localizes to the extracellular compartment either as a secreted protein or as the extracellular domain of a single-pass transmembrane protein. Both Gli and NL are type I membrane proteins that bear a C-terminal PDZ recognition motif, while Nrt is a type II membrane protein. The percentage identity to human AChE is shown in each extracellular domain. Dm stands for Drosophila melanogaster and h stands for human. The hAChE scale bar represents 100 amino acids.

NLs are expressed in both neurons and glia in the nervous system, as well as in a variety of other tissues 3,4,5. At synapses, they are associated with the postsynaptic membrane, and have been shown to bind through their AChE-like domains to the extracellular domains of both α- and β-neurexin, which are also synaptic adhesion proteins 3,6,7. The cytoplasmic domains of the NLs, as well as that of Gli, that is, NL-cyt and Gli-cyt, contain PDZ recognition motifs at their C-termini 2. Indeed, the intracellular partners of the NLs that have been reported are PDZ domain proteins that are known to be associated with the postsynaptic density, such as PSD95 and S-SCAM 8,9. Both PSD95 and S-SCAM have been shown to induce clustering of potassium channels and NMDA receptors 8,10. Whereas it has been shown that NL1 is localized at the postsynaptic membranes of excitatory glutamatergic synapses 11, NL2 is exclusively localized at inhibitory synapses 12. Scheiffele et al., who engineered nonneuronal cells to express NLs, demonstrated their functional importance; the nonneuronal cells induced morphological and functional presynaptic differentiation upon contacting axons in vitro. Moreover, this activity was inhibited by addition of a soluble form of β-neurexin 13. These findings suggest that NLs are involved in formation and remodeling of CNS synapses. Recently, it was shown that a 32 amino-acid sequence in the cytoplasmic domain of NL1 is the dendritic targeting motif of this protein 14,15.

Mutations in the extracellular region of either NL3 or NL4 have been described in certain families with a high frequency of autism spectrum disorders or of X-linked mental retardation 16,17,18. It is plausible that the mutations lead to subtle changes in neuronal circuitry, since certain such mutations have been shown to result in partial intracellular retention of the mutant proteins 19,20. Recently the R451C mutation in NL3 was introduced into mice resulting in impaired social interactions and enhanced spatial learning abilities that, unexpectedly, were accompanied by increased inhibitory synaptic transmission 21.

We earlier showed that the cytoplasmic domain of Gli, Gli-cyt, is natively unfolded 22. Natively unfolded proteins, also known as intrinsically unstructured proteins (IUPs), are flexible, extended, and have little or no ordered secondary structure in vitro in the absence of partners 23. These proteins question the known structure-function paradigm in proteins; despite their lack of a well-defined three-dimensional structure, they are indeed functional 24,25. A combination of low sequence complexity, low overall hydrophobicity, and relatively high net charge are important prerequisites for a protein to be devoid of compact structure 23,26,27. Functionally, IUPs are seen to be involved in protein-protein interaction networks and protein-DNA complexes, and participate in signal transduction, transcriptional activation, nucleic acid recognition, and cell cycle regulation 25. Protein disorder has been suggested to increase the rates of protein association 28. Moreover, evidence has been presented that IUPs display the capacity to interact with multiple protein partners, and thus to function as hub proteins in interactomes (for a recent review see 29), and this binding promiscuity has been suggested to increase network complexity 30. Recently, a number of in silico predictors for protein disorder have been developed which reveal a much higher frequency of IUPs, or of long unfolded stretches, in eukaryotes than in prokaryotes 31. Some of them appear to be associated with scaffolding elements in complex structures such as synapses, which are absent in prokaryotes.

The high flexibility of the IUPs renders them highly sensitive to proteases, a feature that has been taken advantage of in their characterization 32. Recently, we have described an assay based on the susceptibility of IUPs to 20S proteasomal degradation machinery as a simple, rapid, and reliable method of operationally defining IUPs and unstructured sequences 33.

Although IUPs and denatured proteins lack stable secondary structure elements, the left-handed poly-L-proline II helix (PPII) has been found to be prominent in both 34. The main chain dihedral angles in PPII are ϕ,ψ≈−78°, +146°, corresponding to three residues per turn. Its helical rise per residue is 3.1Å, compared to 1.5Å for the α-helix, and it is thus much more extended 35. While proline residues are common in PPII helices, their presence is not obligatory 36. Although proline possesses the highest intrinsic propensity to adopt the PPII conformation, glutamine, alanine, and glycine also display high propensities 37. Already in 1968, Tiffany and Krimm 38 noted a resemblance between the circular dichroism (CD) spectra of denatured proteins and those of PPII and collagen, and suggested that the unfolded state produced by denaturation is, in fact, composed of short stretches (4–7 residues) of PPII connected by sharp bends. Zagrovic et al. 39 have suggested that in the unfolded state, individual residues are likely to sample a broad basin around the PPII region of the Ramachandran map, but do not form regular PPII helices due to the occurrence of rapid changes in their ϕ- and ψ-angles (see also 40).

It was already pointed out by Steinberg et al. 41 that PPII itself is considerably more compact than would be expected for a fully extended rod, and this has been borne out by more recent experimental and theoretical studies 39,42. Thus, it may be expected that IUPs, in general, will be substantially more compact than denatured proteins, as was shown to be the case for Gli-cyt 22 and in other cases 43.

In the following, we describe the overexpression, purification, and physicochemical characterization of the cytoplasmic domain of human neuroligin 3 (hNL3-cyt), and provide evidence that it belongs to the family of IUPs, with a conformation that is not, however, fully extended.

Disorder in hNL3-cyt was assessed using Foldindex 44, the VL-XT algorithm in PONDR 45,46, and IUPred 47. Secondary structure prediction made use of the program PROF 48.

The domain mean net charge 〈R〉, i.e., the net charge at pH 7.0 divided by the number of amino acids, and the domain mean hydrophobicity 〈H〉, i.e., the sum of the normalized hydrophobicities of all residues divided by the number of residues in a given polypeptide sequence, were calculated as described by Uversky et al. 26. The boundary mean hydrophobicity 〈H〉_b, namely, the mean hydrophobicity below which a polypeptide with a given mean net charge 〈R〉 is predicted to be unfolded, was calculated according to Uversky 23 and Oldfield et al. 49.

DNA encoding the intracellular domain of hNL3-cyt (amino acids 731–848) was amplified by PCR from a cDNA template of a full-length human NL3 clone (clone ID 5258980) provided by Dr. Peter Scheiffele (Department of Cell Biology, College of Physicians and Surgeons, Columbia University, New York) using primers with NdeI and NotI restriction sites. The PCR product was purified, restricted with NdeI and NotI, and ligated between the NdeI and NotI sites of the pET28a (Novagen, Madison, WI) expression vector. The sequence of the hNL3-cyt coding region was confirmed by nucleotide sequencing.

Escherichia coli BLR(DE3) cells transformed with hNL3-cyt were grown to saturation overnight at 37°C with shaking in LB medium containing kanamycin (30μg/ml) and tetracycline (12.5μg/ml). An aliquot from the overnight culture was diluted 1:100 in fresh LB medium containing kanamycin (30μg/ml) and tetracycline (12.5μg/ml), and grown at 37°C, with shaking, to OD₆₀₀=0.6, at which time hNL3-cyt expression was induced by addition of isopropyl-β-D-galactoside to a final concentration of 1mM. After a further 3h incubation the cells were harvested by centrifugation (10,000g, 10min, 4°C), yielding ∼2.2g/L of cell pellet, and stored at −80°C. hNL3-cyt labeled with ¹³C and ¹⁵N was produced in transformed E. coli BLR(DE3) cells grown to saturation overnight, at 37°C with shaking, in LB medium containing kanamycin (30μg/ml) and tetracycline (12.5μg/ml). An aliquot from the overnight culture was diluted 1:100 in fresh M9 medium containing kanamycin (30μg/ml) and tetracycline (12.5μg/ml), 6g/l Na₂HPO₄, 3g/l KH₂PO₄, 1g/l ¹⁵N NH₄Cl (Cambridge Isotope Laboratories, Andover, MA), 0.5g/l NaCl, 1ml/l 0.1M CaCl₂, 2ml/l 1M MgSO₄, 2g/l ¹³C D-glucose (Cambridge Isotope Laboratories), 5mg/l thiamine-HCl, 1ml/l trace elements (1mM Cu[OAc], ZnSO₄, MnSO₄, CoSO₄, Ni[SO₄]₂, Na₂MoO₄), and 10ml/l RPMI 1640 vitamin cocktail (Sigma-Aldrich, St. Louis, MO). After incubation with shaking at 225 RPM for 9h at 37°C, hNL-3cyt expression was induced with 1mM isopropyl-β-D-galactoside, and incubation continued overnight until harvesting.

The bacterial cell pellet was resuspended in 30ml 8M urea/100mM NaH₂PO₄/10mM Tris, pH 8.0 (buffer A), clarified by centrifugation (12,000g, 30min, 4°C), and filtered through a 0.22μm Stericup filtration device (Millipore, Billerica, MA). The filtered protein solution was loaded onto a 1ml HisTrap column (GE Healthcare, Waukesha, WI), preequilibrated in buffer A, and eluted using a linear pH 8.0–4.5 gradient of buffer A. Eluted fractions were analyzed by SDS-PAGE, and fractions containing hNL3-cyt were pooled and loaded onto a SP FF cation exchange column (GE Healthcare). The protein was eluted using a linear gradient of 0–1M NaCl in 100mM MES, pH 6.0. Pure fractions were pooled and concentrated, with concomitant change of the buffer to 1mM EDTA/5mM dithiothreitol (DTT)/250mM NaCl/100mM Tris, pH 8.5, using a Vivaspin concentration device (3000 MWCO, Sartorius, Goettingen, Germany), and further purified through a HiLoad 16/60 Superdex 75pg column (GE Healthcare) preequilibrated with the same buffer.

Samples of hNL3-cyt (25μg in a volume of 200μl) and of purified native Torpedo californica AChE (TcAChE) (50μg in a volume of 200μl), in 50mM NaCl/100mM Tris, pH 8.0, were digested at room-temperature by addition of proteinase K (Sigma) to a final concentration of 50ng/ml for hNL3-cyt, and 100ng/ml for TcAChE. Aliquots were removed at appropriate times, mixed with Laemmli sample buffer containing 1mM phenylmethylsulfonyl fluoride (Sigma), and boiled for 5min to arrest proteolysis. Samples were loaded onto a 15% SDS-PAGE gel, electrophoresed, and stained with GelCode.

A 50-μl aliquot of a 10mg/ml solution of purified hNL3-cyt, in 1mM EDTA/5mM DTT/250mM NaCl/100mM Tris, pH 8.5, was injected onto an analytical Superdex 75 HR 10/30column using an AKTA fast-performance liquid chromatography system (GE Healthcare) under both nondenaturing (1mM EDTA/5mM DTT/250mM NaCl/100mM Tris, pH 8.5) and denaturing (8M urea/10mM Tris/100mM NaH₂PO₄, pH 7.0) conditions. The column was calibrated with molecular weight standards under both nondenaturing and denaturing conditions, and a calibration curve was created for the nondenaturing run using Eq. (1) and a plot of K_av versus log molecular weight (see Supplementary Material, Fig. S1, in Data S1 ).

(1)

Fluorescence correlation spectroscopy (FCS) measurements were performed using 20–40nM hNL3-cyt that had been labeled on Cys⁷⁷⁵, the only cysteine residue in the cytoplasmic domain, with ATTO 488 maleimide (ATTO-TEC, Siegen, Germany) according to the manufacturer's protocol. The final step of removal of free label utilized a 1ml HiTrap desalting column (GE Healthcare) in 50mM NaCl/50mM NaH₂PO₄, pH 7.0, on an AKTA FPLC system. Measurements were performed using a home-built confocal microscope, with a water immersion UplanApo 60× NA 1.2 objective (Olympus, Tokyo, Japan). The setup consisted of a 488nm Ar⁺ ion laser (35 LAP 431, Melles Griot, Carlsbad, CA), focused through the objective, at a power level of 30μW. Emitted fluorescence was collected through the objective, filtered by a 500 DCLP dichroic mirror (Chroma, Brattleboro, VT) and an HQ 500 LP long-pass interference filter (Chroma), and then focused by the microscope tube lens onto a 50μm pinhole. The beam was then split by a nonpolarizing beam splitter, and focused onto two identical SPCM-AQR-15 single-photon counting avalanche photodiodes (Perkin Elmer, Fremont, CA). A Flex02-12D/C hardware correlator (Correlator.com, Bridgewater, NJ) was used for data collection and for generation of the FCS curves. The correlation functions were fitted to a model of diffusion through a three-dimensional Gaussian observation volume which takes into account triplet state photodynamics (Eq. (2)) 50.

(2)

Sedimentation velocity experiments were carried out on an Optima XL-A analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) in an An-50Ti rotor. The hNL3-cyt sample was dialyzed in phosphate-buffered saline (PBS) adjusted to pH 8.0, and 400-μl aliquots of the hNL3-cyt sample and of PBS buffer were injected into a double-sector cell with a 12mm Epon centerpiece and quartz windows.

Ultracentrifugation was performed at 50,000rpm, 20°C (after a 3-h temperature equilibration period), at a protein concentration of 0.3mg/ml. The absorbance wavelength was 230nm. A radial step size of 0.003 was used for scanning, and >500 scans were collected, at which point sedimentation was complete.

Data from the first 500 scans were analyzed using the SEDFIT program 51, either with a diffusion deconvoluted sedimentation coefficient c(s) distribution model, with a continuous molar mass distribution c(M), or with the two-dimensional c(s,f_r) approach, which is more suitable for dynamic ensembles of molecules for which the assumption of a continuous f_r in the c(s) distribution is unclear 52. For both approaches the solvent density, the partial specific volume, and the Stokes radius (R_H) were obtained using the SEDNTERP program 53.

Synchrotron x-ray scattering (SAXS) data for hNL3-cyt were collected on the X33 beamline, at the EMBL Hamburg Outstation 54, using a MAR345 image plate detector. The scattering patterns were measured with a 2-min exposure time for multiple solute concentrations ranging from 3 to 1.5mg/ml. DTT was added to a final concentration of 7mM before measurement. To check for radiation damage, two 2-min exposures were compared, and no apparent damage was detected. Using the sample-detector distance of 2.7m, a range of momentum transfer of 0.09<s<0.5Å⁻¹ was covered (s=4πsin(θ)/λ, where 2θ is the scattering angle, and λ=1.5Å is the x-ray wavelength). The data were processed using standard procedures, and extrapolated to infinite dilution using the PRIMUS program package 55. The forward scattering, I(0), and the radius of gyration, R_g, were evaluated using the Guinier approximation 56, assuming that at very small angles (s<1.3/R_g) the intensity is represented as I(s)=I(0)exp(−(sR_g)²/3. Values of I(0) and R_g, as well as the maximum dimension, D_max, and the interatomic distance distribution functions, (p(r)), were computed using the GNOM indirect transform package 57.

The molecular mass of hNL3-cyt was evaluated by comparison of the forward scattering with that for a reference solution of bovine serum albumin (BSA) (66kDa).

To quantitatively characterize the range of conformations adopted by hNL3-cyt in solution, the program EOM was used 58. Given a pool of randomly generated structures (with a R_g distribution similar to that of a random coil), EOM employs a genetic algorithm to select an ensemble of (typically 20–50) models, average scattering from which fits the experimental pattern. The properties of the selected ensembles obtained in multiple EOM runs provide information about the flexibility of the protein in solution.

CD spectra were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK). Far-UV CD spectra were recorded in a 0.1cm path-length cuvette at a protein concentration of 12μM (0.19mg/ml). All CD spectra were recorded in 10mM potassium phosphate, pH 7.0, except for the SDS-containing samples, which were measured in 10mM Tris, pH 7.9. The step size was 0.5nm, and the averaging time 3s. Each trace displayed represents the average of three spectra, and is corrected for the contribution of the buffer. Denaturation experiments utilized high purity guanidinium hydrochloride (Gdn.HCl) (Pierce, Rockford, IL) and trifluoroethanol (TFE) (Sigma). The TFE was handled using glass tips and glass tubes.

1,2-Dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) liposomes (Avanti Polar Lipids, Alabaster, AL) were prepared by evaporation, in a glass test tube, of a 100-μl aliquot of a 20mg/ml chloroform solution, followed by resuspension in 500μl of 10mM KPO₄, pH 8.0, followed by vortexing for 1min, and sonication for 3min at 188W in a Vibracell Sonicator (Sonics, Newtown, CT), to yield a 5mM stock.

The spin-labeled thiol reagent (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methathiosulfinate (MTSSL, Toronto Research Chemicals, Toronto, Ontario, Canada), was used to covalently label the single free cysteine residue, Cys⁷⁷⁵. MTSSL was dissolved in ethanol, and mixed at a 20:1 molar ratio with 200μM hNL3-cyt in 10mM Na₂HPO₄, pH 7.0. After incubation for 14h at 4°C, the reaction mixture was loaded onto a 1ml HiTrap desalting column (GE Healthcare) equilibrated with 150mM NaCl/10mM NaH₂PO₄, pH 7.0, on an AKTA FPLC system, to separate the labeled protein from the free reagent. All measurements were performed on an ELEXSYS 500 spectrometer (Bruker, Billerica, MA), equipped with an Euroterm ER 4113VT temperature unit (Bruker) with an accuracy of ±1K, in a 60μl flat cell, in a protein concentration range of 4–8μM.

The rotational correlation time (τ_R) of weakly immobilized probes was estimated using the equation 59

(3)

NMR samples contained 1mM of uniformly labeled (>95%) ¹⁵N/¹³C-labeled protein in 90%/10% v/v H₂O/D₂O with 5mM dithiothreitol (DTT), 20mM potassium phosphate buffer, pH 6.0, and a trace amount of sodium azide. Experiments were performed at 25.0±0.1°C on a Unity Inova 600MHz spectrometer (Varian, Palo Alto, CA) equipped with a triple resonance probe-head and single-axis pulsed-field gradient capabilities. ¹⁵N Nuclear Overhauser Effect (NOE) spectra were recorded as described by Farrow et al. 60. Two datasets were acquired; one without (reference), and one with proton saturation (steady-state NOE) during the recycle delay. In the reference experiment, the interscan delay was 8s to allow for the complete recovery of proton and nitrogen magnetization. The saturation experiment consisted of a 5s delay followed by 3s of proton saturation by a train of nonselective 120° pulses spaced by 5ms. Reference and saturation experiments were recorded as complete two-dimensional datasets accumulating eight scans per increment in 7h. Five copies of the reference and saturation experiments were acquired, and were merged after data processing to increase the signal/noise ratio while, at the same time, avoiding the accumulation of systematic errors due to long-term spectrometer instabilities. Spectra were recorded with spectral widths of 8000Hz, sampled over 512 complex points in the ¹H dimension (maximum evolution time 64ms), and 1650Hz, sampled over 200 complex points in the ¹⁵N dimension (maximum evolution time 121.2ms). Data sets were processed using NMRPipe 61. The time domain data were apodized using a shifted sine bell in both the ¹H and ¹⁵N dimensions. The final size of each matrix was 1024×512 real points after zero filling and Fourier transformation, giving a digital resolution of 7.8Hz (¹H) and 3.2Hz (¹⁵N), respectively. Complete chemical shift assignments for hNL3-cyt are not yet available.

Determination of mass and peptide mapping, making use of matrix-assisted laser desorption/ionization time-of flight, were performed by the Weizmann Institute Mass Spectrometry Service.

Dynamic light scattering (DLS) measurements were performed on a model No. 802 machine (Viscotek, Houston, TX), in 1mM EDTA/5mM DTT/250mM NaCl/100mM Tris, pH 8.5, and in 6M urea dissolved in the same buffer, at a protein concentration of 1mg/ml.

Hydrodynamic radii to be anticipated for a 15.4kDa protein in various states, e.g., the native globular state, the premolten globule, and under conditions of denaturation at high concentrations of either urea or Gdn.HCl, were calculated according to Uversky 23.

Calculation of the radius of gyration for an unfolded protein was performed using Flory's scaling law 62

(4)

Calculation of the radius of gyration of an unfolded protein was also performed according to the equation utilized by Moncoq et al. 64,

(5)

Calculation of the radius of gyration for a freely jointed chain 67 was performed using

(6)

hNL3-cyt has a low content of hydrophobic amino acids, ∼16.5% aliphatic residues (Val, Leu, and Ile), and ∼4% aromatic amino acids (Phe, Trp, and Tyr). It contains ∼20% charged residues, and has a net charge of −1. Analysis of its sequence, making use of PONDR and Foldindex as predictors of intrinsic disorder, indicated that it contains substantial stretches of both ordered and disordered sequences, although the assignments made by the two programs differ substantially. Both assign the N-terminal 50 amino-acid sequence as disordered (Figure 2AB). However, Foldindex predicts that residues 51–118 will be almost completely ordered, whereas PONDR predicts alternating folded and unfolded sequences. In contrast, IUPred, a predictor that estimates an energy resulting from interresidue interactions 47, predicts the entire hNL3-cyt sequence to be fully unstructured (Figure 2C). In support of this latter assignment, the secondary structure predictor, PROF, predicts that ∼86% of the domain is in loop conformations, with only ∼9% being assigned as two short α-helices, and ∼4% as two β-strands (Figure 2D). Furthermore, three out of four of these motifs have low confidence levels. The overall charge and hydrophobicity values for hNL3-cyt are 〈R〉=0.008 and 〈H〉=0.4295. The boundary mean hydrophobicity 〈H〉_boundary, i.e., the mean hydrophobicity below which a polypeptide with a given mean net charge 〈R〉 will most probably be unfolded 26, is 〈H〉_b=0.407 for hNL3-cyt. Consequently, hNL3-cyt, although actually on the folded side, lies very close to the boundary between the folded and unfolded regions of the charge-hydrophobicity phase space defined by Uversky and co-workers (Oldfield et al. 49). If the two segments, 1–54 and 55–118, are similarly analyzed using the Uversky method, the first segment is predicted to be well within the unfolded phase-space (〈R〉=0.0377, 〈H〉=0.3634, 〈H〉_b=0.418), and the second within the folded region (〈R〉=0.0153 〈H〉=0.4825 〈H〉_b=0.41).

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Figure 2

Bioinformatics analysis of hNL3-cyt. (A) Foldindex prediction; (B) PONDR prediction. Both algorithms predict that the 50 N-terminal amino acids of hNL3-cyt are disordered; (C) IUPred prediction for hNL3-cyt; and (D) PROF secondary structure prediction (in the PROF_sec line, H denotes helix and E denotes strand). Rel_sec line is the reliability index, ranging from 0 (low) to 9 (high).

SDS-PAGE performed on an induced cell pellet of E. coli transfected with hNL3-cyt revealed overexpression of a ∼20kDa protein. This band yielded a positive Western blot with anti-His antibodies (data not shown). MALDI/MS of the band yielded a mass of 15,367Da, the calculated mass for the construct being 15,378Da. Peptide mapping confirmed that the band corresponded to hNL3-cyt, with 11% coverage. Various purification protocols under nondenaturing conditions, even in the presence of a broad-spectrum protease inhibitor cocktail for the purification of His-tagged proteins (Sigma), all resulted in rapid protein degradation (Fig. 3, lane 3). Abnormal migration of the full-length hNL3-cyt domain on SDS-PAGE, as well as CD analysis of the heterogeneous hNL3-cyt solution in the far UV (Fig. S3 in Data S1 ), both provided early indications that hNL3-cyt is an IUP. IUPs are, in general, known to be highly sensitive to proteases 68. We therefore developed a protocol in which purification was carried out under denaturing conditions, which resulted in inactivation of the endogenous E. coli proteases. This approach succeeded, and allowed us to routinely obtain ∼10mg of stable purified NL3-cyt per L of medium (see Materials and Methods, and Fig. 3, lane 2). Such preparations were used in the physicochemical studies described below.

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Figure 3

SDS-PAGE of partially purified hNL3-cyt extracted under denaturing or nondenaturing conditions. hNL3-cyt was extracted from the bacterial pellet either under denaturing conditions (8M urea/100mM NaH₂PO₄/10mM Tris, pH 8.0) or nondenaturing conditions (500mM NaCl/50mM NaH₂PO₄/10mM imidazole, pH 8.0, containing a broad-range protease inhibitor cocktail). In both cases, extraction was at 4°C. Both extracts underwent an initial purification step which involved absorption on a Ni-column followed by elution using a pH gradient for the protein extracted under denaturing conditions, and an imidazole gradient for the sample extracted under nondenaturing conditions (for details, see Materials and Methods). Lane 1, molecular weight markers; Lane 2, hNL3-cyt purified after extraction under denaturing conditions; and Lane 3, hNL3-cyt purified after extraction under nondenaturing conditions. Western blotting, using anti-His antibodies, reveals that many of the fast-moving species below the band of intact hNL3-cyt (arrow) are degradation products (not shown).

As mentioned above, during initial attempts to purify hNL3-cyt under nondenaturing conditions, even in the presence of a broad-range protease inhibitor cocktail, the protein degraded completely over a few days at 4°C, suggesting susceptibility to trace amounts of proteases that had not been completely removed during purification. To confirm this susceptibility to proteolytic digestion, the rate of digestion of hNL3-cyt by Proteinase K was compared to that of TcAChE, a globular protein that is known to be resistant to proteases in its native state 69 (Fig. 4). hNL3-cyt is, indeed, much more sensitive to Proteinase K than native TcAChE.

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Figure 4

Digestion of hNL3-cyt and TcAChE by proteinase K. Digestion conditions were as described under Materials and Methods. (A and B) SDS-PAGE of AChE (A) and hNL3-cyt (B) after digestion by proteinase K. Times of digestion, in minutes, are marked above each lane.

Fig. 5 shows the elution profile of hNL3-cyt under nondenaturing and denaturing conditions. Under denaturing conditions, in 8M urea/100mM NaH₂PO₄/10mM Tris, pH 7.0, the 15kDa protein eluted at a position corresponding to that to be expected for a ∼80kDa globular protein, and to a hydrodynamic radius (R_H) of ∼39Å. In the absence of urea it eluted later, at a position corresponding to that for a ∼41kDa globular protein, and to an R_H of ∼28.4Å, a radius that corresponds to a premolten globule according to the analysis of Uversky 23.

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Figure 5

Size exclusion chromatography of hNL3-cyt. hNL3-cyt eluted at 8.1ml under denaturing conditions (red), and at 9.4ml under nondenaturing conditions (black). A perfect sphere with the molecular weight of hNL3-cyt would be predicted to elute at 11.85ml.

These data show that hNL3-cyt is already substantially unfolded under nondenaturing conditions, but becomes even more extended upon denaturation. Since size-exclusion chromatography (SEC) is not an ideal technique for determining the size and shape of nonglobular proteins, we carried out DLS, FCS, AUC, and SAXS measurements for hNL3-cyt. These techniques provided us with detailed information on its dimensions, and provided further evidence for the presence of a dimer.

The correlation curve for hNL3-cyt under denaturing conditions (6M Gdn.HCl) is shifted to longer diffusion times due to the higher viscosity of the Gdn.HCl solution and the expansion of the polypeptide chain relative to nondenaturing conditions (Fig. 6). Hydrodynamic radii of the nondenatured and denatured forms of hNL3-cyt, calculated using the Stokes-Einstein equation, were 25.0±0.6Å and 33.0±0.8Å, respectively. These values once again demonstrate that hNL3-cyt is disordered in solution, but can be further extended by denaturation.

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Figure 6

Fluorescence correlation curves for ATTO-488 labeled hNL3-cyt. Measurements in 0.01% Tween/50mM NaCl/50mM sodium phosphate, pH 7.0 (■). Measurements in 0.01% Tween/6M Gdn.HCl/12.5mM NaCl/12.5mM sodium phosphate, pH 7.0 (●). The green solid lines represent fits of the data to the model in Eq. (2). Residuals of data fits are displayed at the bottom of the figure for both the two correlation curves and for the fits.

Analysis of hNL3-cyt fractions eluted from the preparative Superdex 75column, using SDS-PAGE gels at high protein concentrations, and Western blots, displayed the presence of several minor oligomer bands (Fig. 7). To quantify this phenomenon, and to characterize the ensemble of different conformations that hNL3-cyt adopts in solution, we utilized analytical sedimentation velocity ultracentrifugation. The data points and fits that were used in the continuous c(s) distribution are shown in Figure 8A. The c(s) distribution (Figure 8B) exhibits a major peak at 1.33S, with a shoulder at ∼2S. Using Sedfit, it can be calculated that a perfect sphere with the mass of hNL3-cyt should have an S value of 2.15. Thus, it is plausible that the 1.33 S peak corresponds to an extended monomer, and that the shoulder corresponds to a dimer, though it cannot be excluded that it may, in fact, be a collapsed monomer. To evaluate this, a c(s,f_r) analysis, which uses different friction coefficients for different populations, was applied (Figure 8C). If the ∼2 S population is a spherical monomer, it should have a friction coefficient that approaches a value of 1. Both the 1.33 and 2 S populations have high values of f_r, ∼1.8 and 1.5, respectively, which supports the assignment of the shoulder as an extended dimer. The c(M) conversion (Figure 8D) clearly shows that the principal species is a monomer with MW=15,227±300Da, in agreement with the calculated weight of hNL3-cyt, and R_H=26Å, calculated by use of SEDNTERP, taking the value of 1.33S obtained in the c(s) analysis.

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Figure 7

SDS-PAGE and Western-blot of hNL3-cyt after nondenaturing size exclusion chromatography. hNL3-cyt elutes in several oligomeric states, which are stable under the conditions of SDS-PAGE. Lanes 1 and 2, SDS-PAGE of hNL3-cyt loaded at 2.4mg/ml, and 4.8mg/ml; lane 3, Western-blot of hNL3-cyt using anti-His antibody, which displays at least three distinct oligomeric states.

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Figure 8

Analytical ultracentrifugation of hNL3-cyt. An AUC sedimentation velocity experiment was run at 0.3mg/ml in PBS adjusted to pH 8.0. (A) Sedimentation data (solid line) and c(s) analysis fit (dotted line) of every 10th scan. Residuals are shown at the bottom, with a root mean-square deviation of 0.022. (B) c(s) distribution of hNL3-cyt. hNL3-cyt has a broad distribution with a principal peak (S=1.33), and a shoulder (∼2S). (C) Plot of S versus the frictional ratio, f/f_o, derived from the c(s,f_r) analysis. Both populations of hNL3-cyt have high f/f_o values, indicating that both are extended in solution. (D) c(M) distribution of the same data. The first peak has a maximum at 15,227±300Da, in excellent agreement with the molecular weight predicted from the sequence.

The scattering patterns for hNL3-cyt under nondenaturing conditions (1mM EDTA/7mM DTT/250mM NaCl/100mM Tris, pH 8.5) and under denaturing conditions (4M urea/1mM EDTA/7mM DTT/250mM NaCl/100mM Tris, pH 8.5) are shown in Figure 9A. The processed scattering patterns, for hNL3-cyt under both denaturing and nondenaturing conditions, yield an effective molecular mass of ∼14kDa, in agreement with the value predicted from the sequence, indicating that the protein was mostly monomeric in solution. The corresponding distance distribution functions, p(r), are shown in Figure 9B. Under denaturing conditions, hNL3-cyt populates the extended conformations more than under nondenaturing conditions. The values of the radius of gyration (R_g) derived from Guinier's law and the p(r) functions for hNL3-cyt are 30 and 33Å, respectively, under nondenaturing conditions, and 37 and 40Å under denaturing conditions, using both methods. The Kratky plot (s²(I) vs. s) is commonly used to distinguish between compact and extended protein conformations. hNL3-cyt displays a shape typical of unfolded proteins, i.e., a plateau that increases monotonically at higher s values, whereas that for BSA displays a bell-shaped Kratky plot (Figure 9C). The R_g distribution of the structures selected by EOM (Figure 9D, black curve) for hNL3-cyt under nondenaturing conditions displays a bimodal profile. The major component adopts a more compact conformation than a modeled random coil (blue curve), with an average R_g=27Å, compared to 33Å for the random coil. But a second peak, at R_g=53Å, may correspond either to a population of highly extended molecules or the presence of dimers and higher-order oligomers. This bimodal distribution is an attribute of the native protein only; upon addition of urea the reconstructed ensembles display a monomodal R_g profile (Figure 9D, red curve), which more closely resembles that of the random coil distribution (R_g=35Å).

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Figure 9

SAXS measurements on hNL3-cyt. (A) X-ray scattering patterns of hNL3-cyt under denaturing (●) and nondenaturing conditions (■). The fits of the ensembles selected by EOM are displayed as gray lines. (B) Distance distribution functions of the measurements shown in panel A. (C) Kratky plots (red and black traces) derived from the data in panel A and of the globular protein, BSA (blue trace), which served as a folded control. (D) Radii of gyration distributions of hNL3-cyt under denaturing (red line) and nondenaturing conditions (black line) and comparison with a pool of randomly generated structures (blue line).

Circular dichroism (CD) in the far UV was used to assess the conformation of hNL3-cyt and the effects induced by denaturants and other reagents (Fig. 10). In 10mM potassium phosphate, pH 8.0, the CD spectrum has a broad minimum at ∼200nm, which shifts to slightly higher wavelengths, with a concomitant decrease in negative ellipticity, as the temperature is raised (Figure 10A). There is an isodichroic point at 207nm, above which wavelength the negative ellipticity actually increases as the temperature is raised. All three spectra lack any major contributions from α-helices, β-sheets or turns, and are characteristic of an unstructured polypeptide, with a possible PPII contribution 36,70,71.

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Figure 10

CD spectra of hNL3-cyt. All spectra were collected at an hNL3-cyt concentration of 12μM with the appropriate buffer corrections. (A) CD spectra of hNL3-cyt in 10mM potassium phosphate (KPi), pH 8.0, at various temperatures (solid line, 5°C; dash-dotted line, 25°C; and dotted line, 45°C). (B) Effect of 7M Gdn.HCl and temperature on the CD spectrum of hNL3-cyt (solid line, 10mM KPi, pH 8.0, at 5°C; long-dash, short-dash line, −7M Gdn.HCl at 5°C; dash-dotted line, 7M Gdn.HCl at 25°C; and dotted line, 7M Gdn.HCl at 45°C). (C) Effect of 50% TFE and temperature on the CD spectrum of hNL3-cyt (solid line, 10mM KPi, pH 8.0, at 5°C; long-dash, short-dash line, 50% TFE at 5°C; dotted line, 50% TFE at 25°C; and dash-dotted line, 50% TFE at 45°C). (D) Effect of 0.5mM SDS and 1mM DOPS liposomes on the CD spectrum of hNL3-cyt (solid line, 10mM KPi, pH 8.0, 25°C; long-dash, short-dash line, 0.5mM SDS; and dotted line, 1mM DOPS).

It has been suggested that chemical denaturants increase PPII content of unfolded proteins by bonding to the polypeptide backbone, thus shifting the PPII↔disordered equilibrium to the left 72. In line with this observation, Figure 10B shows that, at 5°C, in 7M Gdn.HCl, a maximum appears at ∼224nm, which has been ascribed to PPII 73; this peak becomes slightly negative at 25°C, and even more negative at 45°C. Unfortunately, the presence of Gdn.HCl precludes corresponding measurements at wavelengths <220nm. Measurements with urea gave similar results (data not shown). TFE is known to promote α-helix formation in long polypeptides and proteins 74. Indeed, 50% TFE converts the CD spectrum of hNL3-cyt at 25°C to that of a more helical polypeptide, a conformation which is maintained even at 45°C (Figure 10C).

In situ, the cytoplasmic domain of NL is adjacent to the cell membrane. Possible structural changes induced by the bilayer were mimicked in vitro by addition of SDS at submicellar concentrations of 0.5 and 3mM, or by 1mM DOPS liposomes, also resulting in a small shift to a more helical conformation (Figure 10D). The zwitterionic detergent CHAPS, at 0.45% w/v, caused no shift in the CD spectrum of hNL3-cyt, nor did the crowding agents Ficoll 70 (30mg/ml), sucrose (1M), and the osmoprotectant betaine (4M) (data not shown).

The shape of the ESR spectrum provides a measure of the mobility of the bound nitroxyl radical, which depends on the structure and flexibility of the protein in the vicinity of its site of attachment. The ESR signal of spin-labeled hNL3-cyt in 150mM NaCl/10mM sodium phosphate, pH 7.0, at 20°C reveals relatively sharp peaks (Figure 11A), indicating only limited restriction of the probe's movement. When a 100-fold excess of DTT is added (Figure 11B), these relatively slow components disappear completely, with concomitant appearance of a sharp triplet, characteristic of unrestricted rotation of the free radical. This provides clear evidence that the probe had indeed formed a mixed disulfide with the free thiol group of the protein, which had then been reduced by DTT, with concomitant release of the spin label. Figure 11C shows the ESR spectrum obtained in the presence of 66% TFE. The peak intensity of the high-field component (h_H) of the ESR spectrum in the conjugate decreases relative to that seen in the absence of TFE, indicating a decrease in segmental mobility of the polypeptide.

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Figure 11

ESR measurements on spin-labeled hNL3-cyt. (A) The ESR spectrum of spin-labeled hNlg3-cyt (in 150mM NaCl/10mM sodium phosphate, pH 7.0, at 20°C), reveals relatively sharp peaks that indicate rather limited restriction of the probe's mobility. (B) Disappearance of the relatively slow components, with concomitant appearance of a sharp triplet, when a 100-fold molar excess of DTT was added to the spin-labeled protein. (C) ESR spectrum obtained in the presence of 66% TFE, at 20°C. The peak intensity of the high-field component (h_H) in the conjugate (which is very sensitive to label movement) decreases relative to that seen in the absence of TFE, suggesting a decrease in segmental mobility of the polypeptide. (D) Temperature-dependence of the (h_C)/(h_H) ratio. Both in the absence (■) and presence of TFE (▴), the ratio decreases with temperature, reflecting an increase in mobility.

Rotational correlation times for the bound nitroxyl radical were calculated according to Eq. (3), yielding τ_R values of 9.3×10⁻¹⁰ and 1.3×10⁻⁹s in buffer and TFE, respectively. Comparison of these values to that for isotropic rotation of the free radical in solution, τ_R∼10⁻¹¹−10⁻¹⁰s, and to values for folded proteins, τ_R>10⁻⁷s, indicates limited restriction of the motion of the bound radical in buffer, and more substantial restriction in TFE.

Figure 11D shows the temperature dependence of the ratio of the peak intensity of the central component (h_C) to that of h_H, which, as mentioned above, reflects the mobility of the covalently bound spin label. Both in the absence and presence of TFE, the ratio decreases with temperature, reflecting increased mobility. The decrease seen in the presence of TFE is more pronounced, presumably since the starting conformation is more structured. However, in both cases, no sharp transition is seen in the range of 20–65°C.

NMR spectra recorded for hNL3-cyt are typical of an unfolded protein: The signals are narrow and display very limited dispersion in the proton dimension. As can be seen in Figure 12A, the backbone amide proton chemical shifts are restricted to the coil region, i.e., 7.7–8.7ppm. Signals observed at 112/7.0ppm (¹⁵N/¹H) originate from arginine side chains, whereas the cross peak at 130/10ppm stems from the side chain indole of the sole tryptophan. Although the ¹⁵N-¹H 2D NMR spectra of Figure 12AB, have the appearance of an HSQC spectrum, they were recorded with the heteronuclear ¹⁵N{¹H} NOE pulse sequence 60. The spectrum obtained with ¹H irradiation during the ¹⁵N relaxation delay (Figure 12B) is dramatically different from the reference experiment (Figure 12A). The signal attenuation by dipolar cross relaxation (NOE) is so strong that it leads to negative crosspeaks. Such behavior is typical of unfolded proteins 75, whereas attenuation factors of 15% would be expected for the ordered regions in a folded protein with a molecular weight of 15kDa. The extent of signal attenuation can be gleaned more clearly from Figure 12CD, which were taken at 120ppm from Figure 12AB, respectively. The uniform attenuation observed for all the signals indicates that persistent structure cannot be present for any portion of the polypeptide chain on timescales longer than several nanoseconds, and that the dynamics of the amide backbone are dominated by segmental flexibility. The arginine side chains and the last residues of the sequence have greater motional freedom than backbone amides more central to the chain, and therefore give rise to even stronger negative NOEs.

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Figure 12

NMR measurements on hNL3-cyt. Contour plots of two-dimensional ¹⁵N{¹H} NOE NMR spectra recorded without (A) and with (B) proton saturation. Spectra are shown at the same contour level. Contour levels in spectrum A are positive, and in panel B are negative. Arg NE denotes side-chain N^ɛ-H^ɛ correlations that are aliased in the ¹⁵N dimension from ∼84.7ppm. One-dimensional traces taken from panels A and B at 120ppm (¹⁵N) are shown in panels C and D, respectively.

The cytoplasmic domains of the CLAMs, Gli-cyt, Nrt-cyt, and NL-cyt, display no sequence homology to any other polypeptide sequences or indeed to each other, except that Gli-cyt and NL-cyt possess tetrapeptide PDZ recognition motifs at their C-termini 2, and a few common eukaryotic linear motifs 76 characteristic of either phosphorylation sites or WW domain interaction sites. It is worth noting that sequence conservation among the cytoplasmic domains of the NLs is ∼31%, compared to ∼55% for their corresponding folded extracellular ChE-like domains.

We earlier presented physicochemical evidence, complemented by bioinformatic analysis, that Gli-cyt is an IUP, i.e., that the purified protein domain adopts an unstructured conformation in aqueous solution under physiological conditions 22. In this study, we have carried out similar characterization of hNL3-cyt.

While bioinformatic analysis clearly predicted that Gli-cyt, with a net charge of +7 and ∼15% hydrophobic amino acids, should be an IUP, as was indeed confirmed by its physicochemical characterization, the situation with respect to NL3-cyt is somewhat ambiguous. As described under Results, hNL3-cyt has ∼20% hydrophobic amino acids, and a net charge of −1. Furthermore, the first 54 amino acids, which have the same net charge as the whole sequence, have only ∼14% hydrophobic residues; consequently, making use of the method of Uversky and co-workers 49, this segment is predicted to be unfolded (〈R〉=0.0377, 〈H〉=0.3634, 〈H〉_b=0.418). Residues 55–118 have zero net charge and ∼29% hydrophobic residues; thus, making use of the same method this segment is predicted to be folded (〈R〉=0.0153 〈H〉=0.4825 〈H〉_b=0.41). The same analysis of the entire sequence predicts that the whole protein will fall just on the folded side of the boundary. Evidence that IUPs may contain regions that are predicted to be folded has been presented for such proteins as α-synuclein, negative factor, and helix-destabilizing protein 26. It has been suggested that the regions of these proteins predicted to be intrinsically unfolded prevent the remainder of the sequence from folding, perhaps through electrostatic interactions.

When PONDR and Foldindex are used to analyze the hNL3-cyt sequence, both clearly predict that the 50 NH₂-terminal residues are unfolded, but Foldindex predicts that residues 54–118 should be folded, whereas PONDR predicts alternating folded and unfolded stretches. Yet, secondary structure predictors predict minimal amounts of secondary structure. Furthermore, IUPred predicts the whole domain to be unstructured. Taking into account these predictions, hNL3-cyt might be assigned to the class of dynamic and collapsed IUPs, with some secondary structure propensity, as defined by Uversky and co-workers 49. These somewhat contradictory findings illustrate the pitfalls of bioinformatics analysis, and clearly show the need for experimental clarification. Although this study focused on hNL3-cyt, it is worth mentioning that the cytoplasmic domains of all the NLs have similar patterns in the various secondary structure and unstructured predictors.

Bearing in mind the ambiguities in the bioinformatics analysis we decided to clone, express, and purify hNL3-cyt, so as to permit its physicochemical characterization.

As mentioned above, hNL3-cyt was rapidly degraded in the course of purification. This was a positive indication that hNL3-cyt is indeed an IUP, since unstructured proteins are highly sensitive to proteolysis, a feature that has been widely used as an experimental tool for characterizing IUPs 32. Purification under denaturing conditions was used to deactivate the proteases present during the initial stages, with a transition to nondenaturing buffers during the final purification steps. We suggest this method as a general procedure for purification of IUPs. It could thus serve as an alternative to other more aggressive lysis and purification techniques, such as boiling 77, which might result in protein aggregation and concomitant trapping of IUPs in the aggregates.

hNL3-cyt is relatively unstable in solution, and displays a tendency to aggregate reversibly at high concentrations (>5mg/ml), upon contact with chemical chaperones and positive micelles, and during various mechanical manipulations, all of which hinder certain experiments. A possible explanation for this instability is the presence of hydrophobic residues in hNL3-cyt that are not part of an intramolecular hydrophobic core, and, thus being exposed, have a propensity to participate in intermolecular interactions.

Obtaining a relatively stable form of hNL3-cyt enabled us to carry out structural characterization under a variety of conditions using an extensive repertoire of methodologies.

Several physicochemical techniques, e.g., FCS, SDS-PAGE 78, SEC, SAXS, NMR, all provide strong evidence that hNL3-cyt has an extended conformation, as summarized in Table 1. Both CD in the far UV and NMR, that displays limited backbone amide chemical shift dispersion, rule out the presence of substantial amounts of classical secondary structure, in agreement with the prediction of PROF. Instead of retaining a fixed secondary structure, the protein rapidly interconverts between different conformations of similar free energy. The attenuation obtained in the heteronuclear ¹⁵N{¹H} NOE NMR experiment shows that the chain is flexible on the (sub)nanosecond timescale, indicating that energy barriers between the visited conformations are small. Furthermore, there is no significant ellipticity in the near UV, little binding of ANS, no change in tryptophan fluorescence, and only a slight increase in mobility of the already mobile spin label upon denaturation (data not shown). These data, taken together, suggest that hNL3-cyt is an IUP in vitro, in the size range of a premolten globule 79,80 as operationally defined by Uversky 23. Indeed, FCS, SAXS, and SEC clearly show that it becomes substantially more extended under denaturing conditions under which it approaches dimensions expected for a random coil configuration (see Table 1). The more compact dimensions of hNL3-cyt under nondenaturing conditions than in the presence of denaturants may be ascribed to dynamic intrachain interactions that constrict some parts of the polypeptide chain in water. It is possible that its conformation may bear some resemblance to the collapsed conformations observed during protein folding from denaturant 81 as reported for the intrinsically unfolded NM segment of the yeast prion protein, Sup35 82.

The ESR measurements provide local information pertaining to the vicinity of the bound radical, and clearly show that this region is highly flexible in solution. Thermal denaturation experiments monitored using both ESR and CD (data not shown) reveal a monotonic change in conformation with temperature, with no evidence for a two-state transition typical of globular proteins 83.

Near-UV CD can, in principle, serve as a tool to detect constrained conformational freedom of aromatic side chains in solution, but its utility for studying IUPs is rather limited. This is because, in general, IUPs have a low aromatic content 84, as well as a low level of nonlocal interactions which, in turn, might favor fast interconversions of those interactions which exist. Breaking of these intrachain interactions by denaturants should thus produce a more extended structure. It has, however, been suggested that denaturants increase the percentage of residues in the PPII conformation relative to disordered backbone conformations 73,85. Since PPII is believed to consist of short stretches of residues in an extended helical conformation, broken by sharp turns, a polypeptide sequence with a high and stable PPII content should, in principle, have more expanded dimensions than a true random coil 86. Thus, if a protein containing the same number of residues as hNL3-cyt were assumed to be entirely composed of PPII helices, with a persistence length of 4nm (corresponding to ∼14 residues) 41,42, crudely modeled for simplicity as a freely jointed chain 67, the calculated radius of gyration would be ∼52Å, as compared to ∼40Å calculated for a random coil by two different methods (Table 1). None of the experimental values that we measure, even under strongly denaturing conditions, approach the value for a PPII jointed chain, but values corresponding to a random coil are attained. This does not exclude the possibility that sequences with PPII propensity may exist, possibly with shorter persistence lengths which might, moreover, dynamically fluctuate along the chain.

As mentioned above, it has been proposed that increasing denaturant concentration produces increased PPII content in short peptides and proteins 73. In these terms of reference, protein denaturation can be considered as a transition from a globally structured state to a dynamic ensemble of locally structured states. Denaturation of IUPs cannot, however, be viewed in such terms. Rather, their denaturation can be seen as a transition from a set of globally flexible, extended, and largely unstructured states to a dynamic ensemble of even more extended locally structured states. The CD spectrum of a PPII helix consisting entirely of proline residues displays a dichroic minimum at ∼200nm, and a maximum at 228nm, which shifts toward lower wavelengths if the polypeptide contains a substantial percentage of residues other than proline 87. Upon heating, the ∼200nm minimum becomes less pronounced, and the ellipticity at ∼228nm decreases, suggesting that the PPII↔unstructured equilibrium is shifted to the right due to destruction of the water shell around the polypeptide that lacks intrachain hydrogen bonds which might otherwise serve to stabilize it 38,88. The aforementioned ellipticity maximum characteristic of PPII is absent from the spectrum of hNL3-cyt under nondenaturing conditions, but small increases in ellipticity at 228nm are observed for hNL3-cyt upon denaturation with either Gdn.HCl or urea (Figure 10B), implying that denaturation raises the propensity of hNL3-cyt to assume a PPII conformation. It may be noted that a much more pronounced effect is observed in similar measurements performed on Gli-cyt (A. Paz, J. L. Sussman, and I. Silman, unpublished).

Taken together, the hydrodynamic and spectroscopic data discussed above suggest that the PPII conformation may contribute to the ensemble of conformations that an IUP samples, but that in hNL3-cyt there are apparently no prominent PPII helices per se.

An alternative interpretation of the spectral changes observed, in particular of the reversible changes in ellipticity at 200 and 222nm observed upon heating, is the turnout response; a gain in structural complexity due to strengthening of the hydrophobic effect, resulting in a stronger driving force for partial folding 23,89,90.

We chose to refrain from quantifying the secondary structure content on the basis of the far-UV CD spectra, since use of the various algorithms and reference data sets implemented in DichroWeb 91 yielded conflicting results. The main reason for these inconsistencies is the lack of reliable reference data sets for IUPs. Furthermore, although ellipticity at 222nm (θ₂₂₂) has been widely employed as a measure of α-helical content, both in globular proteins and in IUPs 92, this same parameter has also been used to estimate PPII content 93,94. Indeed, Bienkiewicz et al. 95 used this criterion to estimate PPII content of an IUP, assuming a two-state equilibrium between PPII and unstructured conformations. However, if an IUP were to possess some α-helical content, it would affect the two-state assumption, and, in any case, it would not be possible to apply the θ₂₂₂ measure to assessment of both PPII and α-helical content simultaneously. Thus, caution should be exercised in utilizing far-UV CD for quantification of the structural motifs present in a given IUP that might contain various residual structure elements along its polypeptide chain, such as molecular recognition features 96, that would contribute differently to θ₂₂₂.

Having established that hNL3-cyt is an IUP puts previous studies on NL into a new perspective. hNL3 is a human homolog of Drosophila Gli, based on sequence homology of their extracellular AChE-like domains 4. However, their intracellular domains display no detectable sequence homology, except for the short linear motifs referred to in the Introduction. Nevertheless, both are IUPs that are expressed at cellular junctions and appear to participate in protein-protein interactions. Investigation of the evolutionary rates of IUPs revealed that, in general, they are less conserved than ordered proteins 97. If a domain's disorder is important to its interaction with multiple partners, and hence to its plastic function, it may well be that there is no evolutionary pressure to maintain a particular amino acid, as long as it is replaced by an amino acid which is also disorder-promoting, so that mutation does not bring about an increase in secondary structure in the disordered domain. In such a case, the only specific evolutionary pressure for sequence preservation in the cytoplasmic CLAM domains will apply to the four-residue PDZ recognition motif at the COOH-terminus, and other putative recognition motifs along the sequence, thus explaining the sequence divergence within the CLAM family. It has, furthermore, been noted that the cytoplasmic domains of the various NLs are less conserved than their extracellular ChE domains (31% and 55% identity, respectively); the sequence differences between the various NL-cyt domains was attributed to localization signals or to other unknown functions 98. We raise the possibility that this low degree of sequence conservation can be ascribed to the aforementioned weaker evolutionary pressure on the disordered cytoplasmic sequences.

NL has been shown to putatively bind 13 proteins via interactions through its PDZ recognition motif 9. This promiscuity was further suggested by the authors to be enhanced by the promiscuous binding of the target proteins themselves to additional multiple partners. We suggest that the complexity of the interactome of NL-cyt is further amplified due to its unstructured character; consequently, it may have the capacity to bind to even more targets in a PDZ-domain-independent manner. In this context, it may be noted that a large percentage of hub proteins in interactomes are, indeed, IUPs 29,99,100,101,102,103. The degree of sequence homology of the cytoplasmic domains of the human NLs argues that they will function similarly in an intracellular context.

A major issue in research on IUPs is their conformational state in vivo. For some IUPs, experimental evidence suggests that they are fully unfolded within the living cell 104. For a number of other IUPs, it has been shown that binding to a partner such as a protein or DNA is coupled to folding, and has been termed induced folding 24. hNL3-cyt has the propensity to form helices upon addition of TFE. More biologically relevant is the moderate structuring induced in hNL3-cyt in the presence of either SDS or DOPS liposomes, which may mimic the physiological environment of NLs.

The high sensitivity of IUPs to proteases offers the possibility of tight regulation of their expression pattern 33; indeed, disorder has recently been shown to be correlated with short half-lives 105. A number of proteins have been found to bind to NL, some of which colocalize with it within the postsynaptic density 9. We have previously shown that binding of PSD-95 and/or S-SCAM to hNL3-cyt can protect it from degradation by the 20S proteasome 33. Obviously, under conditions of development and plasticity, changes in expression patterns of partner proteins could render hNL3-cyt and its homologs readily susceptible to degradation.

In this study we have shown that hNL3-cyt is an IUP, like Gli-cyt (and most likely also the cytoplasmic domain of a third CLAM, neurotactin 2). As already mentioned, IUPs break the accepted structure/function paradigm that folding to a single native conformation confers biological function and specificity 106. On the contrary, IUPs have the potential to assume multiple conformations, and to interact with multiple partners. Indeed, many IUPs have been shown to serve as hub proteins in interactomes 99. The synapse is a site at which functional flexibility, i.e., synaptic plasticity, is believed to play crucial roles in development and remodeling of the nervous system, in particular in learning and memory 107. We have shown that hNL3-cyt is an IUP, whose structural flexibility confers on it functional promiscuity that is in keeping with the roles assigned to NLs in synaptic assembly 108.