| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 T.C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218, USA
2 Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine and Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA
3 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA
Reprint requests to: Doug Barrick, T.C. Jenkins Department of Biophysics, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA; e-mail: barrick{at}jhu.edu; fax: (410) 516-4118.
(RECEIVED June 26, 2003; FINAL REVISION August 1, 2003; ACCEPTED August 6, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03279003.
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Keywords: Notch signal transduction; ankyrin repeats; signaling mutants; X-ray crystallography; protein stability
Abbreviations: RMSD, root mean square deviation • CD, circular dichroism spectroscopy
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
1000 residues in Drosophila melanogaster) is cleaved from the membrane and enters the nucleus, where it can participate in transcriptional activation of downstream targets (Jarriault et al. 1995; Schroeter et al. 1998; Struhl and Greenwald 1999). Notch signaling activity is modulated by interaction between the intracellular portion of the Notch receptor and a number of cytosolic and nuclear effector proteins, such as Suppressor of Hairless (Matsuno et al. 1997), Deltex (Diederich et al. 1994; Matsuno et al. 1995), EMB-5 (Hubbard et al. 1996), and Skip (Zhou et al. 2000). Many of these interactions appear to involve direct binding to a set of ankyrin repeats in the cytosolic portion of the Notch receptor. As would be expected of a region that facilitates multiple binding reactions, the Notch ankyrin repeats have been shown to be critical for Notch signaling (Rebay et al. 1993; Roehl and Kimble 1993), and a number of mutations that introduce residue substitutions within the Notch ankyrin domain disrupt Notch signaling (Kodoyianni et al. 1992; Diederich et al. 1994; Kopan et al. 1994; Joutel et al. 1996; Kurooka et al. 1998).
Ankyrin repeats are 33 residues in length and consist of two 
-helices connected by a short loop. Adjacent ankyrin repeats are linked together via a long loop that terminates with a tight ß-turn (Gorina and Pavletich 1996; Luh et al. 1997; Batchelor et al. 1998; Huxford et al. 1998; Jacobs and Harrison 1998; Russo et al. 1998; Venkataramani et al. 1998; Foord et al. 1999; Mandiyan et al. 1999; Sedgwick and Smerdon 1999). The sequence conservation among the seven ankyrin repeat sequences of the Notch ankyrin domain is modest (17% average pairwise identity; Zweifel and Barrick 2001a); however the sequence conservation of analogous repeats from different taxa is quite high (approximately 70% identity, see Fig. 1
and Stifani et al. 1992). The Drosophila Notch receptor contains six tandem ankyrin sequence repeats previously identified as closely matching the ankyrin consensus sequence (Bork 1993) and a putative seventh (C-terminal) repeat that exhibits lower similarity to the consensus (Zweifel and Barrick 2001a). Modeling the seventh repeat sequence as a C-terminal ankyrin repeat suggests that residues deviating from the consensus sequence would be exposed to solvent, providing a possible explanation for the lack of conservation of these residues (Zweifel and Barrick 2001a). A polypeptide containing all seven repeats was found to have a free energy of unfolding nearly twice that of a polypeptide lacking the C-terminal repeat (Zweifel and Barrick 2001b), suggesting that this putative seventh ankyrin repeat is an integral part of the Notch ankyrin domain.
|
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
). The overall architecture of the domain consists of an elongated array of antiparallel -helices, consistent with an ankyrin repeat structure. Moreover, the seventh C-terminal ankyrin repeat of all three chains clearly adopts an ankyrin fold (repeat seven, violet; Fig. 2A), consistent with previous thermodynamic and solution structural studies (Zweifel and Barrick 2001a,b). The charged and polar residues in repeat seven that deviate from the ankyrin consensus are solvent exposed (Fig. 2B; compare Arg 221, Asp 222, and Ser 225 of repeat seven with the analogous residues Leu 188, Phe 189, and Ala 192 of repeat six). In contrast, the N-terminal sequence repeat (repeat one, red; Fig. 2A) does not adopt a regular ankyrin fold. This repeat is variably disordered among the three chains in the asymmetric unit, and shows significant conformational heterogeneity when the three chains are superposed over repeats two through seven (Fig. 3A). Only the C-terminal portion of the first repeat of each chain is visible in the electron density (from residues 29, 36, and 47 in chains A, B, and C, respectively; see Fig. 1
for numbering), and it adopts a different conformation in each of the three chains.
|
|
|
). Repeats two through seven superpose with an average pairwise root mean square deviation (RMSD) of 0.17 Å (over 184 C atoms of each of the three polypeptides in the asymmetric unit). This close structural similarity is significantly higher than that among individual 33 residue Notch ankyrin repeats, which superpose with an average pairwise RMSD on C of 0.60 Å (Fig. 3B).
The structural similarity between the three Notch ankyrin polypeptides of the asymmetric unit is also significantly higher than that between the Notch ankyrin domain and ankyrin domains from other proteins. Using the program VAST (Madej et al. 1995), the highest structural similarity between the Notch ankyrin domain and another ankyrin structure, obtained by aligning repeats three through seven of the Notch ankyrin domain with the five ankyrin repeats of GABPß (Batchelor et al. 1998), produces an RMSD of 1.1 Å over 149 C atoms (Fig. 3C). The longest alignment, involving the six repeats of I
B and the Notch ankyrin domain (Jacobs and Harrison 1998), produces an RMSD of 1.9 Å over 188 C atoms (Fig. 3C). These comparisons indicate that although the tertiary structures of different ankyrin repeat domains show significant variability, tertiary structure is precisely specified by amino acid sequence in spite of the modular, linear architecture of these domains.
Contacts between ankyrin repeat polypeptides in the crystal lattice
Although our previous hydrodynamic studies at pH 8.0 indicate that the Notch ankyrin domain is monomeric, two-hybrid and other indirect interaction studies have suggested a direct homotypic interaction involving the ankyrin domain of the Notch receptor (Roehl et al. 1996; Matsuno et al. 1997; Kurooka et al. 1998). To identify potential modes by which the Notch ankyrin domain may self-associate, perhaps with the assistance of specific dimerization partners in trans, we examined interactions between Notch ankyrin polypeptides within the crystal lattice.
Although most interchain contacts within the crystal lattice appear to be rather modest, we find one extensive interface, represented three times among the three polypeptides of the asymmetric unit. Within the crystal lattice, each polypeptide in the asymmetric unit is related to its neighbors by a noncrystallographic threefold screw axis that runs perpendicular to the long axis of each ankyrin domain. This threefold screw is continued by the adjacent polypeptide of the next unit cell. Along this screw axis, each polypeptide forms two substantial pseudosymmetric interfaces, one each with each of its two nearest neighbors. Interchain contacts are made between repeats two, three, and four on the convex surface of one polypeptide and repeats four, five, and six of the concave surface of the neighboring polypeptide (Fig. 4A). Each interface buries roughly 1500 Å2 of total surface area per contact (750 Å2 per polypeptide) and yields a relatively high shape correlation parameter (Lawrence and Colman 1993; average Sc = 0.74 for the three interfaces). This shape correlation value, which is a measure of the proximity and the shape similarity of contacting surfaces, is as large as values for stable, well-characterized protein oligomer and protease/protein inhibitor complexes (Lawrence and Colman 1993).
|
). At one edge of each interface (near repeat four of the convex surface), a feature in the electron density is seen that appears to be a magnesium ion, with octahedral coordination to five waters and a single aspartic acid (Asp 136) from the convex surface of repeat four. The residues surrounding this magnesium ion include several acidic residues; thus this region has a high negative electrostatic potential (Fig. 4B,C). The presence of this divalent ion may be required to neutralize the negative charge that is juxtaposed at this interface, consistent with the high (200 mM) magnesium concentrations required for crystallization.
To test whether stable homomeric complexes form at lower pH values (the nominal pH during crystallization was 6.8, whereas our previous hydrodynamic studies were done at pH 8.0) or are stabilized by magnesium ions, we performed multi-angle static light scattering over a range of pH values in the presence and absence of magnesium. At 150 mM NaCl, the Notch ankyrin domain remained monomeric over the examined pH range (6.5–8.0), both in the absence and presence of MgCl2 (Fig. 5, Table 2). Thus, the interface observed within the lattice alone is not sufficient to promote tight association of the Notch ankyrin domain.
|
|
), and thus can be mapped onto the structure of the Drosophila Notch ankyrin domain with reasonable certainty (Fig. 6).
|
), indicating that these surface substitutions do not greatly perturb the Notch ankyrin fold. In contrast, the spectra of proteins containing substitutions within the core of the domain (M1 and M2) differ significantly from that of the parent construct, showing a substantial loss of -helical structure.
|
). The two surface substitutions (Su42c and F18) have only modest effects on the unfolding transitions. In contrast, substitutions that include buried residues (M1 and M2) result in major shifts in the unfolding transitions to lower urea concentrations. In addition, the observed structural transitions of the M1 and M2 variants are less cooperative, and appear to be underway in the absence of urea. In terms of unfolding free energy, the surface substitutions (Su42c and F18) have only minor effects on unfolding free energy, whereas the substitutions that include buried residues (M1 and M2) decrease unfolding free energy by 7–8 kcal mole-1 (Fig. 7C). Thus, the Su42c and F18 substitutions are likely to disrupt signaling by directly perturbing interactions with specific components of the pathway, whereas the M1 and M2 substitutions are likely to disrupt the overall fold of the ankyrin domain, indirectly disrupting interactions with multiple interacting partners in a nonspecific manner. |
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
In contrast, the first ankyrin sequence repeat appears to be largely disordered. The visible portion of this repeat adopts a different conformation in each of the three polypeptides present in the asymmetric unit. This heterogeneity is consistent with the observation that a core mutation in the first Notch ankyrin sequence repeat has very little effect on stability, whereas analogous mutations in repeats two through seven are highly destabilizing (Bradley and Barrick 2002). It is unlikely that the lack of order in the first repeat can be attributed to N-terminal sequence outside the ankyrin domain that is absent from our construct, because inclusion of additional N-terminal sequence (135 residues) enhances neither helix formation nor stability of the Notch ankyrin domain (M.E. Zweifel and D. Barrick, unpubl.). Furthermore, the elimination of the N-terminal sequence that extends between the Notch transmembrane and the ankyrin repeat domains does not alter the observed homotypic association of the Notch ankyrin domain as determined using a yeast two-hybrid assay (Matsuno et al. 1997).
Although the two helices of the first sequence repeat show a close match to the ankyrin consensus sequence, an obvious difference between the first sequence repeat and the other ankyrin repeats is that there is an insertion of 15 residues between the two consensus helical regions of the first repeat (Fig. 1), whereas the two helical segments are connected by a short two-residue turn in other ankyrin sequences. This insertion may contribute to the instability and/or heterogeneity of the first sequence repeat, either through strain or through an entropic penalty for loop closure. Similar insertions are seen in the first sequence repeat of Notch ankyrin domains from a range of species (Fig. 1, see + signs). Although these inserted sequences vary in length, they have a high proportion of acidic (D, E) and polar (S, T, N, G) residues. This conservation suggests that the insertion may play a functional role in Notch signaling, and may undergo a functionally important disorder–order transition upon the binding of one or more effector proteins, as has been seen in several other systems (Daughdrill et al. 1997; Dyson and Wright 2002). One functional role for this acidic sequence may be in transcriptional activation (Giniger and Ptashne 1987; Gill and Ptashne 1988), given that the intracellular region of the Notch receptor acts as a cotranscription factor in the activated state.
Potential interactions between ankyrin repeat polypeptides
Indirect interaction studies have suggested a homotypic interaction of the Notch receptor involving the ankyrin domain (Roehl et al. 1996; Matsuno et al. 1997; Kurooka et al. 1998). The extensive contact surface identified between each of the three polypeptides of the asymmetric unit are consistent with such an interaction, although association via the crystallographically observed interface in solution would be expected to lead the formation of helical fibers, rather than to a closed complex of discrete stoichiometry. Despite this large crystallographic interface, we find the Notch ankyrin domain to remain monomeric under conditions where it is soluble from pH 6.5 to 8.0, in 150 mM NaCl. Furthermore, the Notch ankyrin domain remains monomeric under these conditions in the presence of high MgCl2 concentrations, indicating that the interfacial magnesium ion seen in the crystal structure is not, on its own, sufficient to mediate oligomerization under the conditions examined here. Thus, if this mode of interaction is relevant in solution, it must be rather weak, and may be enhanced by secondary binding either to effector proteins or through indirect interactions with DNA. One system in which weak association plays a role in function is the regulation of transcription in Escherichia coli by GalR. Although GalR is largely dimeric in solution (Majumdar et al. 1987), it is thought to mediate DNA looping through weak tetramerization, facilitated by HU protein (Semsey et al. 2002). Weak binding interactions have the potential to enhance allosteric responses, maximizing the degree to which populations of various noncovalent complexes can be modulated by changes in the concentrations of the polypeptides from which they are formed.
Stability of Notch ankyrin domain variants
We have examined all reported mutations that can be mapped to the Drosophila Notch ankyrin domain. Analysis of the effect of these substitutions on stability reveals that the two single-residue substitutions (Su42c and F18) located at surface-exposed positions do not significantly perturb the overall stability of the domain, whereas the two multiresidue substitutions (M1 and M2) that include one or more buried positions greatly diminish the overall stability of the domain, resulting in significant disruption of structure. Given the location of these substitutions within the structure of the Notch ankyrin domain and their effects on stability, the substitutions that include buried positions (M1 and M2) seem likely to block Notch signaling activities through general disruption of the fold of the Notch ankyrin domain, whereas the surface substitutions (Su42c and F18) seem likely to block signaling through disruption of direct interactions with a limited set of effector proteins.
Although not directly comparable, the severity of the effects of these substitutions on Notch signaling is consistent with the effects of these substitutions on the structural stability of the Notch ankyrin domain. Consistent with this proposal, the Su42c mutation is phenotypically similar to mutations in the Deltex gene, the product of which has been shown to interact with the Notch ankyrin domain in a two-hybrid interaction assay (Diederich et al. 1994). Thus, this surface substitution may interfere with the Notch–Deltex interaction. Su42c, a surface substitution that has only a minor effect on stability, produces relatively mild, nonlethal changes in the adult body plan (Diederich et al. 1994). Similarly, the effects of the F18 surface substitution, which also has only minor effects on stability, do not appear until relatively late in adult life (Joutel et al. 1996). The location of these two surface substitutions may define the site of interaction with specific effectors of Notch signaling. In contrast, mutations that result in core substitutions (M1 and M2) abolish MyoD/RBP-J-mediated transcriptional activity in cultured cells, a central activity in the Notch signaling pathway (Kopan et al. 1994). Although the effects of these multisite substitutions on normal development have not been determined, disruption of Notch-mediated transcriptional activation would likely produce severe developmental defects. Disruption of signaling as a result of substitutions at buried positions within an ankyrin domain has also been seen in the tumor suppressor p16INK4A, for which structurally disruptive substitutions have been identified in several cancer cell lines (Tevelev et al. 1996; Tang et al. 1999).
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
[Bradley and Barrick 2002] encoding residues 1902–2139 of the Drosophila Notch receptor) was expressed in E. coli strain BL21(DE3) as described previously (Zweifel and Barrick 2001a). Selenomethionine was incorporated by expression in E. coli strain B834(DE3) (a methionine auxotroph) grown in minimal media supplemented with seleno-L-methionine. Protein was purified from the soluble fraction of a bacterial lysate using a Ni•NTA affinity column. The protein was then dialyzed into buffer containing 150 mM sodium chloride and 25 mM Tris-HCl (pH 8.0) and then treated with thrombin to remove the 6xHis-tag. The protein was further purified by ion-exchange and gel-filtration chromatography, concentrated to 10–15 mg/ml, and dialyzed into buffer containing 150 mM sodium chloride and 25 mM Tris-HCl (pH 8.0). Crystals were grown by hanging drop vapor diffusion with 15%–20% (v/v) MPD, 200 mM magnesium acetate, and 100 mM sodium cacodylate (pH 6.5–7.0). Crystals grew in 1 to 2 weeks to a size of approximately 0.1 x 0.1 x 0.05mm, belonging to the tetragonal space group P43212, with a = b = 73.6 and c = 341.1 Å.
Structure determination and refinement
Crystals were soaked in a cryoprotectant solution consisting of 25% (v/v) MPD, 200 mM magnesium acetate, and 100 mM sodium cacodylate at pH 7.0 for 30 sec and then flash-frozen at -175°C. A 2.8 Å native data set was collected at this temperature using a Rigaku RU-300HR X-ray generator with an R-AxisIV detector. Crystals diffracted to a resolution of 2.0 Å; however, at resolutions higher than 2.8 Å, reflections could not be resolved because of the long (c = 341.1 Å) axis of the unit cell. Diffraction data from the selenomethionine derivative was collected at National Synchrotron Light Source (NSLS) beamline X-4A and processed using DENZO/SCALEPACK (Otwinowski and Minor 1997). Initial phases were calculated using SOLVE/RESOLVE (Terwilliger and Berendzen 1999) to produce an interpretable map. An initial model was constructed using the program O (Jones et al. 1991). Refinement of the initial model against the 2.8 Å native data set was performed using CNS (Brunger et al. 1998). To extend resolution, a second native data set (2.0 Å) was collected at NSLS beamline X-25 to improve separation of higher resolution reflections. The 2.8 Å model was refined against the 2.0 Å native data set using REFMAC (Murshudov et al. 1997) to Rworking = 18.0 and Rfree = 20.1. The final model contained 5042 atoms including 471 solvent molecules. The quality of the final model was assessed using the program PROCHECK (Laskowski et al. 1993). Over 90% of residues were found to be in the most favored regions of the Ramachandran plot, and showed good stereochemistry (Table 1). Buried surface area was calculated using the method of Lee and Richards (1971) as implemented in the program CNS (Brunger et al. 1998). The shape correlation parameter was calculated using the program SC (Lawrence and Colman 1993) from the CCP4 suite (Collaborative Computational Project 1994).
Measurement of the conformational stability of Notch mutants
Notch mutants were constructed by site-directed mutagenesis using the QuikChange Kit (Stratagene, La Jolla, CA). Mutations were verified by sequencing and were purified as described previously (Zweifel and Barrick 2001a). Unfolding free energies were estimated by urea denaturation in 25 mM Tris-HCl, 150 mM NaCl (pH 8.0) at 20°C. Unfolding transitions were detected by monitoring the CD signal at 222 nm using an Aviv model 62DS CD spectrometer as described (Zweifel and Barrick 2001b). Free energies of unfolding were extrapolated to zero denaturant concentration assuming a linear dependence on denaturant (Pace 1986; Santoro and Bolen 1988) as described previously (Zweifel and Barrick 2001b). For the unfolding curves for the M1 and M2 variants, the native CD signal was assumed to be the same as that for the parent construct. This assumption was necessary because these two variants only show partial unfolding transitions. Thermodynamic analysis of partial unfolding transitions of the Notch ankyrin domain has previously been shown to provide reasonable estimates of unfolding free energies (Mello and Barrick 2003).
Static light scattering measurements
Static light scattering measurements were performed at 25°C using an HPLC system equipped with a TosoHaas G3000PWXL HPLC column, a three-angle light scattering detector (Wyatt MiniDAWN; Wyatt Technologies, Santa Barbara, CA) and a differential refractive index detector (Wyatt Optilab DSP; Wyatt Technologies). Samples were dialyzed overnight at 4°C into running buffer (150 mM NaCl, either 25 mM Tris at pH 8.0 or 25 mM HEPES at pH 7.4, 7.0, and 6.5, and 0.01% sodium azide) with or without 10 mM MgCl2. Chromatographic separation was performed at a flow rate of 0.5 ml/min. 50 µl samples were injected onto a preequilibrated column at a loading concentration between 1.5 and 3.0 mg/ml (approximately 60 to 120 µM protein). Data acquisition and analysis were performed using ASTRA 4.0 software (Wyatt Technologies).
Coordinates
The atomic coordinates of the Notch ankyrin domain have been deposited in the Protein Data Bank (PDB ID 1OT8).
|
Acknowledgments |
|---|
. This research was supported by a Beckman Young Investigator award to D.B., and by NIH grant GM60001. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Barton, G.J. 1993. ALSCRIPT—A tool to format multiple sequence alignments. Protein Eng. 6: 37–40.
Batchelor, A.H., Piper, D.E., de la Brousse, F.C., McKnight, S.L., and Wolberger, C. 1998. The structure of GABP/ß: An ETS domain-ankyrin repeat heterodimer bound to DNA. Science 279: 1037–1041.
Bork, P. 1993. Hundreds of ankyrin-like repeats in functionally diverse proteins: Mobile modules that cross phyla horizontally? Proteins 17: 363–374.[CrossRef][Medline]
Bradley, C.M. and Barrick, D. 2002. Limits of cooperativity in a structurally modular protein: Response of the Notch ankyrin domain to analogous alanine substitutions in each repeat. J. Mol. Biol. 324: 373–386.[CrossRef][Medline]
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography and NMR System (CNS): A new software system for macromolecular structure determination. Acta Crystallogr. D 54: 905–921.[CrossRef][Medline]
Collaborative Computational Project. 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50: 760–763.[CrossRef][Medline]
Daughdrill, G., Chadsey, M., Karlinsey, J., Hughes, K., and Dahlquist, F. 1997. The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, sigma 28. Nat. Struct. Biol. 4: 285–291.[CrossRef][Medline]
Diederich, R.J., Matsuno, K., Hing, H., and Artavanis-Tsakonas, S. 1994. Cytosolic interaction between Deltex and Notch ankyrin repeats implicates Deltex in the Notch signaling pathway. Development 120: 473–481.[Abstract]
Dyson, H.J. and Wright, P.E. 2002. Coupling of folding and binding for unstructured proteins. Curr. Opin. Struct. Biol. 12: 54–60.[CrossRef][Medline]
Esnouf, R.M. 1997. An extensively modified version of molscript that includes generally enhanced colouring capabilities. J. Mol. Graphics Modelling 15: 132–134.[CrossRef][Medline]
Foord, R., Taylor, I.A., Sedgwick, S.G., and Smerdon, S.J. 1999. X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function. Nat. Struct. Biol. 6: 157–165.[CrossRef][Medline]
Gill, G. and Ptashne, M. 1988. Negative effect of the transcriptional activator GAL4. Nature 334: 721–724.[CrossRef][Medline]
Giniger, E. and Ptashne, M. 1987. Transcription in yeast activated by a putative amphipathic helix linked to a DNA binding unit. Nature 330: 670–672.[CrossRef][Medline]
Gorina, S. and Pavletich, N.P. 1996. Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science 274: 1001–1005.
Hubbard, E.J., Dong, Q., and Greenwald, I. 1996. Evidence for physical and functional association between EMB-5 and LIN-12 in Caenorhabditis elegans. Science 273: 112–115.[Abstract]
Huxford, T., Huang, D.B., Malek, S., and Ghosh, G. 1998. The crystal structure of the IB/NF-B complex reveals mechanisms of NF-B inactivation. Cell 95: 759–770.[CrossRef][Medline]
Jacobs, M.D. and Harrison, S.C. 1998. Structure of an IB/NF-B complex. Cell 95: 749–758.[CrossRef][Medline]
Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., and Israel, A. 1995. Signalling downstream of activated mammalian Notch. Nature 377: 355–358.[CrossRef][Medline]
Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47: 110–119.
Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E., et al. 1996. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383: 707–710.[CrossRef][Medline]
Kabsch, W. and Sander, C. 1983. Dictionary of protein secondary structure: Pattern recognition of hydrogen bonded and geometrical features. Biopolymers 22: 2577–2637.[CrossRef][Medline]
Kidd, S., Kelley, M.R., and Young, M.W. 1986. Sequence of the Notch locus of Drosophila melanogaster: Relationship of the encoded protein to mammalian clotting and growth factors. Mol. Cell. Biol. 6: 3094–3108.
Kodoyianni, V., Maine, E.M., and Kimble, J. 1992. Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans. Mol. Biol. Cell 3: 1199–1213.[Abstract]
Kopan, R., Nye, J.S., and Weintraub, H. 1994. The intracellular domain of mouse Notch: A constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 120: 2385–2396.
Kraulis, P.J. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystal. 24: 946–950.[CrossRef]
Kurooka, H., Kuroda, K., and Honjo, T. 1998. Roles of the ankyrin repeats and C-terminal region of the mouse Notch1 intracellular region. Nucleic Acids Res. 26: 5448–5455.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. 1993. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystal. 26: 283–291.[CrossRef]
Lawrence, M.C. and Colman, P.M. 1993. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234: 946–950.[CrossRef][Medline]
Lee, B. and Richards, F.M. 1971. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 55: 379–400.[CrossRef][Medline]
Luh, F.Y., Archer, S.J., Domaille, P.J., Smith, B.O., Owen, D., Brotherton, D.H., Raine, A.R., Xu, X., Brizuela, L., Brennner, S.L., et al. 1997. Structure of the cyclin-dependent kinase inhibitor p19Ink4d. Nature 389: 999–1003.[CrossRef][Medline]
Madej, T., Gibrat, J.-F., and Bryant, S.H. 1995. Threading a database of protein cores. Proteins 23: 356–369.[CrossRef][Medline]
Majumdar, A., Rudikoff, S., and Adhya, S. 1987. Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector. J. Biol. Chem. 262: 2326–2331.
Mandiyan, V., Andreev, J., Schlessinger, J., and Hubbard, S.R. 1999. Crystal structure of the ARF-GAP domain and ankyrin repeats of PYK2-associated protein ß. EMBO J. 18: 6890–6898.[CrossRef][Medline]
Matsuno, K., Diederich, R.J., Go, M.J., Blaumueller, C.M., and Artavanis-Tsakonas, S. 1995. Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121: 2633–2644.[Abstract]
Matsuno, K., Go, M.J., Sun, X., Eastman, D.S., and Artavanis-Tsakonas, S. 1997. Suppressor of Hairless-independent events in Notch signaling imply novel pathway elements. Development 124: 4265–4273.[Abstract]
Mello, C.C. and Barrick, D. 2003. Measuring the stability of partly folded proteins using TMAO. Protein Sci. 12: 1522–1529.
Merritt, E.A. and Bacon, D.J. 1997. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277: 505–524.[Medline]
Murshudov, G.N., Vagin, A.A., and Dodson, E.J. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53: 240–255.[CrossRef][Medline]
Nicholls, A., Sharp, K.A., and Honig, B. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281–296.[CrossRef][Medline]
Otwinowski, Z. and Minor, W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307–326.
Pace, C.N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131: 266–280.[Medline]
Rebay, I., Fehon, R.G., and Artavanis-Tsakonas, S. 1993. Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74: 319–329.[CrossRef][Medline]
Roehl, H. and Kimble, J. 1993. Control of cell fate in C. elegans by a GLP-1 peptide consisting primarily of ankyrin repeats. Nature 364: 632–635.[CrossRef][Medline]
Roehl, H., Bosenberg, M., Blelloch, R., and Kimble, J. 1996. Roles of the RAM and ANK domains in signaling by the C. elegans GLP-1 receptor. EMBO J. 15: 7002–7012.[Medline]
Russo, A.A., Tong, L., Lee, J.O., Jeffrey, P.D., and Pavletich, N.P. 1998. Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a. Nature 395: 237–243.[CrossRef][Medline]
Santoro, M.M. and Bolen, D.W. 1988. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27: 8063–8068.[CrossRef][Medline]
Schroeter, E.H., Kisslinger, J.A., and Kopan, R. 1998. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393: 382–386.[CrossRef][Medline]
Sedgwick, S.G. and Smerdon, S.J. 1999. The ankyrin repeat: A diversity of interactions on a common structural framework. Trends Biochem. Sci. 24: 311–316.[CrossRef][Medline]
Semsey, S., Geanacoloulos, M., Lewis, D.E.A., and Adhya, S. 2002. Operator-bound GalR dimers close DNA loops by direct interaction: Tetramerization and inducer binding. EMBO J. 21: 4349–4356.[CrossRef][Medline]
Stifani, S., Blaumueller, C.M., Redhead, N.J., Hill, R.E., and ArtavanisTsakonas, S. 1992. Human homologs of a Drosophila enhancer of split gene product define a novel family of nuclear proteins. Nat. Genet. 2: 119–127.[CrossRef][Medline]
Struhl, G. and Greenwald, I. 1999. Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398: 522–525.
Tang, K.S., Guralnick, B.J., Wang, W.K., Fersht, A.R., and Itzhaki, L.S. 1999. Stability and folding of the tumour suppressor protein p16. J. Mol. Biol. 285: 1869–1886.[CrossRef][Medline]
Terwilliger, T.C. and Berendzen, J. 1999. Automated MAD and MIR structure solution. Acta Crystallogr. D 55: 849–861.[CrossRef][Medline]
Tevelev, A., Byeon, I.J., Selby, T., Ericson, K., Kim, H.J., Kraynov, V., and Tsai, M.D. 1996. Tumor suppressor p16INK4a: Structural characterization of wild-type and mutant proteins by NMR and circular dichroism. Biochemistry 35: 9475–9487.[CrossRef][Medline]
Vardar, D., North, C.L., Sanchez-Irizarry, C., Astor, J.C., and Blacklow, S.C. 2003. Nuclear magetic resonance structure of a prototype Lin12-Notch repeat module from human Notch1. Biochemistry 42: 7061–7067.[CrossRef][Medline]
Venkataramani, R., Swaminathan, K., and Marmorstein, R. 1998. Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nat. Struct. Biol. 5: 74–81.[CrossRef][Medline]
Wharton, K.A., Johansen, K.M., Xu, T., and Artavanis-Tsakonas, S. 1985. Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43: 567–581.[CrossRef][Medline]
Zhou, S., Fujimuro, M., Hsieh, J.J.-D., Chen, L., Miyamoto, A., Weinmaster, G., and Hayward, S.D. 2000. SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC to facilitate NotchIC function. Mol. Cell. Biol. 20: 2400–2410.
Zweifel, M.E. and Barrick, D. 2001a. Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 1. Solution conformational and hydrodynamic properties. Biochemistry 40: 14344–14356.[CrossRef][Medline]
———. 2001b. Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding. Biochemistry 40: 14357–14367.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Hayward, T. Kalmar, and A. Martinez Arias Wnt/Notch signalling and information processing during development Development, February 1, 2008; 135(3): 411 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Niessen and A. Karsan Notch signaling in the developing cardiovascular system Am J Physiol Cell Physiol, July 1, 2007; 293(1): C1 - C11. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. F. Kelly, R. J. Lake, T. Walz, and S. Artavanis-Tsakonas Conformational variability of the intracellular domain of Drosophila Notch and its interaction with Suppressor of Hairless PNAS, June 5, 2007; 104(23): 9591 - 9596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. U. Ferreiro and E. A. Komives The plastic landscape of repeat proteins PNAS, May 8, 2007; 104(19): 7735 - 7736. [Full Text] [PDF]
|
|
|
|
|
|
T. O. Street, C. M. Bradley, and D. Barrick Predicting coupling limits from an experimentally determined energy landscape PNAS, March 20, 2007; 104(12): 4907 - 4912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ehebauer, P. Hayward, and A. Martinez-Arias Notch Signaling Pathway |
