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Anchoring Notch Genetics and Biochemistry
Molecular Cell, Vol. 13, 619–626, March 12, 2004, Copyright 2004 by Cell Press
Anchoring Notch Genetics and Review Biochemistry: Structural Analysis of the Ankyrin Domain Sheds Light on Existing Data Olga Y. Lubman,1 Sergey V. Korolev,2 and Raphael Kopan1,* 1 Department of Molecular Biology and Pharmacology and the Department of Medicine Washington University School of Medicine 2 Edward A. Doisy Department of Biochemistry and Molecular Biology Saint Louis University School of Medicine Saint Louis, Missouri 63104
Notch signaling is important in development and in human disease. Notch receptors regulate transcription through direct interactions with several proteins at the promoter regions of target genes. To understand the mechanism of Notch signaling, numerous deletion and mutagenesis studies have been carried out to identify functional domains in Notch, but domain definition and their role during the assembly of the transcriptionally active complex remains controversial. Recently reported biophysical and structural studies of the Notch ANK domain permit us to reevaluate the existing domain assignments and their predicted functional role, thereby providing further insight into the mechanism of Notch signaling.
Notch is an essential part of an ubiquitous signaling pathway involved in a variety of cell fate decisions in metazoans (Artavanis-Tsakonas et al., 1999; Bray, 1998). Mutations in Notch pathway genes cause several human disorders (Gridley, 2003) and can either promote (Ball and Leach, 2003; Ellisen et al., 1991; Maillard and Pear, 2003; Nam et al., 2002) or suppress (Nicolas et al., 2003) cancer. Since the discovery of the Notch locus by Dexter in 1914, studies of the Notch signaling pathway progressed for eight decades without the scrutiny of structural biology. Over the last decade and most recently in a publication describing the crystal structure of the Notch ankyrin repeats (Zweifel et al., 2003), structural biologists joined their distinctive voice to the chorus of Notch investigators. In this review, we will integrate the insights gained from structural and biophysical studies of the Drosophila Notch ankyrin repeats with those gained by genetic and biochemical experiments in many organisms. It is our view that several domain assignments made previously on the basis of biochemical and genetic experiments will require redefinition in light of this critical new information. The Notch protein is a large (300 kDa) single-pass type I transmembrane receptor activated by a regulated intramembrane proteolysis (RIP; Brown et al., 2000). Defined structural domains in the extracellular domain of
*Correspondence: [email protected]
Notch include EGF (Figure 1, yellow; Rao et al., 1995) and LNR (Lin-Notch repeats, orange; Vardar et al., 2003) repeats. Signaling through Notch is initiated by binding of surface-anchored ligands to EGF-like repeats 11 and 12 (shaded) in the extracellular portion of the receptor. The binding of ligand induces proteolysis that sheds the extracellular portion of the Notch receptor. This is followed by a second cleavage hydrolyzing a peptide bond within the transmembrane domain; as a result, the Notch intracellular domain (NICD) dissociates from the membrane and enters the nucleus (for review see Fortini, 2002; Mumm and Kopan, 2000). Instead of possessing an enzymatic activity, NICD orchestrates changes in DNA-bound protein assemblies in the nucleus, centered around the DNA binding protein CSL [CBP or RBPjk in vertebrates, Su(H), Drosophila, Lag-1 in C. elegans; we will mention specific proteins by name when describing data obtained with that protein]. In the absence of Notch, RBPjk interacts with the proteins SKIP and SMRT to recruit one of several transcriptional corepressor complexes (Zhou et al., 2000; Zhou and Hayward, 2001). Therefore, successful signaling via Notch receptors depends on the ability of NICD to displace the corepressor complex associated with RBPjk and convert it to a transcriptionally active complex. Two domains in NICD involved in its interaction with CSL were defined by several nonbiophysical methods. The first was discovered in a yeast two-hybrid assay; it is called the RAM (RBPjk-associated molecule) domain (Tamura et al., 1995). While some critical amino acids for RAM domain/RBPjk interactions were identified by mutagenesis, the exact boundaries of this domain are poorly defined. The RAM domain spans the region from just beyond the Arg/Lys cluster (serving as stop translocation signal at the cytosolic side of the transmembrane domain) to the first ankyrin repeat (Figure 1, magenta). Defining the exact boundaries of the RAM domain is further complicated by the fact that this region appears to be unstructured as evidenced by CD spectroscopy measurements (Nam et al., 2003). Sequence similarity recognized the second domain as a cluster of ankyrin repeats (ANK domain), a highly conserved motif (Figure 1, blue) responsible for mediating protein interactions (Bork, 1993; Groves and Barford, 1999; Sedgwick and Smerdon, 1999; Wharton et al., 1985). Genetic experiments demonstrated that the ANK domain is essential for all known Notch functions (Artavanis-Tsakonas et al., 1999; Greenwald, 1994). NICD interacts strongly with CSL via the RAM domain with the ANK domain contributing to this interaction (Jarriault et al., 1995; Kato et al., 1997; Kodoyianni et al., 1992; Matsuno et al., 1997; Tani et al., 2001). Once the NICD/CSL complex is formed, the protein Mastermind/SEL-8/LAG-3 (MAM) binds to this complex (Doyle et al., 2000; Nam et al., 2003; Petcherski and Kimble, 2000; Wallberg et al., 2002; Wu et al., 2000). Histone acetyltransferases (HAT) are recruited next (Fryer et al., 2002; Kao et al., 1998; Oswald et al., 2001; Wallberg et al., 2002) to assemble the active transcription complex. The domain thought to mediate direct
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Figure 1. Domain Organization of Notch Receptors This diagram uses mouse Notch1 as a prototype. Proteolytic cleavage by furin at site 1 (S1) produces two subunits, which remain noncovalently associated at the cell surface. EGF-like modules 11 and 12 participate in ligand binding (shaded, EGF repeats in yellow). Two consecutive proteolytic cleavages induced upon ligand activation release the intracellular domain of Notch (NICD). NICD consists of the RAM domain (magenta), NLS (nuclear localization signal), ankyrin repeats (blue), TAD (transactivation domain, black), and STR-Ser/Thr-rich domain regulating cytokine response. PEST (proline, glutamate, serine, threonine-rich) sequence and OPA (glutamine-rich sequence) motifs are located C-terminal to the ankyrin repeats; the former regulates protein turnover while the latter contains an “acidic blob” that is thought to enhance transcriptional activation by Notch proteins. (A) EP domain, which maps just C-terminal to the sixth ankyrin repeat, was defined as the region mediating direct interaction with the HAT (histone acetylases) protein p300 (red). (B) Crystal structure of the Drosophila ANK domain confirmed the presence of the seventh repeat that overlaps with the EP domain. The first “ankyrin repeat” does not adopt a regular ankyrin fold and is shaded; the overall number of ankyrin repeats that adopt a canonical ankyrin fold is six. (C) Ribbon representation of the structure of Drosophila ANK domain (Zweifel et al., 2003). The repeats are numbered consecutively.
interaction between Notch and HAT proteins PCAF and GCN5 was mapped to a region C-terminal to the ANK domain and designated the transactivation domain (TAD; Kurooka and Honjo, 2000). Studies using purified proteins demonstrate that the presence of MAM is essential for the recruitment of the HAT protein p300 (Fryer et al., 2002; Wallberg et al., 2002); others propose that direct interaction between Notch and p300 contributes to the recruitment of p300 to the complex (Kurooka and Honjo, 2000; Oswald et al., 2001; Wallberg et al., 2002). Further dissection of TAD using deletion mutagenesis identified the smaller “EP” domain (included in the RE/ AC domain [Beatus et al., 2001] and at the N-terminal boundary of the TAD domain) as critical for transcription activation, potentially through interaction with p300 (Oswald et al., 2001). The question, which needs to be examined in light of the structural studies, is this: do the amino acids included in the EP domain constitute an additional independent subdomain of Notch, and if so, what are its boundaries and its functional significance? Based on the crystal structure of the Drosophila Notch ANK domain and biophysical experiments, the deletion experiments aimed at understanding the function of this region in Notch may have instead demonstrated (again) the importance of the ANK domain to Notch function.
Newly Defined Boundaries of the Notch ANK Domain The placement of the ANK domain in most published Notch sequences was based on homology with available consensus sequences (Bork, 1993; Breeden and Nasmyth, 1987; Kodoyianni et al., 1992; Wharton et al., 1985). Although in their experimental design most authors considered the intracellular portion of Notch receptors to contain only six ankyrin repeats, some recognized the presence of the seventh ankyrin repeat (Bork, 1993; Wettstein et al., 1997). Evidence supporting the existence of a seventh repeat in the archetypical Drosophila Notch were provided by urea-induced unfolding experiments and other biophysical measurements (Nam et al., 2003; Zweifel and Barrick, 2001a, 2001b) and was unequivocally confirmed by the 2.0 A˚ crystal structure of the ANK domain obtained by Barrick and colleagues (Zweifel et al., 2003). The crystal structure further revised the placement of the repeats because it demonstrated that the first (most N-terminal) ankyrin repeat does not adopt a regular ankyrin fold, although we can not completely rule out that CSL binding might induce substantial conformational changes in this region allowing it to adopt the ankyrin fold conformation. Regardless of whether the Notch intracellular domain has six or seven properly folded ankyrin repeats, the crystal structure has
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Figure 2. Natural and Synthetic Mutations in the Ankyrin Domain of Metazoan Notch-1 Proteins and Their Location Relative to the Crystal Structure Structure-based sequence alignment of the ANK domain of Notch-1 orthologs from six different species: D. melanogaster (Dm; AC# AH005262), M. musculus (Mm; AC# NP032740), H. sapiens (Hs; AC# P46531), X. Laevis (Xl; AC# XP342393), R. norvegicus (Rn; AC# NP077334), and glp-1 from C. elegans (Ce; AC# A32901). The alignment was generated using programs CLUSTALV and ESPcript (ExPASy Molecular Biology Server; www.expasy.org). Identical residues are shown in white on black background and homologous residues are shown on gray background. Note that reduced homology is the consequence of including glp-1 in this alignment. Helices of each repeat from the structure of Drosophila ANK domain (Zweifel et al., 2003) are represented by cylinders above the primary sequences. The boundaries of the TAD and EP domains are shown as blue and orange bars, respectively. The mutations are numbered according to their order of appearance in Table 1 in which their molecular nature, behavior in assays, and impact on structure (when known) are described. Mutations and truncations that completely disrupt Notch functions are highlighted in red, while those with milder phenotype (hypomorphic or temperature sensitive) are highlighted in cyan. The Ala substitution of DI(V)_(V)RL_LDE residues is shown in mutation 3–5; note that Val2099 is mutated in both constructs 3 and 4. Many Notch truncations within this ankyrin repeat were published; colored lines indicate the C-terminal boundary of few selected truncated-Notch constructs used by many investigators in Notch field. The citations in Table 1 credit the labs that created or described the truncation.
redefined the C-terminal boundary of the ANK domain by including an additional repeat (Figure 1). What is the relevance of extending the ANK domain for interpretation of cell-based functional analyses in which part of this region was deleted to define the roles of the EP, RE/AC, and TAD domains? To answer this question, we need to first understand what ankyrin repeats are and to examine the contribution of the seventh ankyrin repeat to the stability and folding of the entire ANK domain. Cooperative Unfolding of Notch Ankyrin Repeats The ankyrin repeat is an array of ␣-helical motifs positioned in tandem that is found in a variety of proteins responsible for mediating protein-protein interactions necessary for transcriptional regulation, cell-cycle control, cytoskeletal organization, and cell differentiation
(Breeden and Nasmyth, 1987). Despite considerable sequence variability, ankyrin repeat-containing proteins show very regular and conserved secondary and tertiary structure. Each repeat consists of approximately 33 residues forming two antiparallel ␣ helices connected by a short loop (Figure 2). The adjacent repeats are packed via hydrophobic interactions and are connected by a  hairpin, extended perpendicular to the helical axes to form an L shape structure (Figure 1; Sedgwick and Smerdon, 1999). The elongated tertiary structure of ankyrin repeatcontaining proteins differs significantly from that seen in typical globular proteins, where multiple residues distant from each other in the primary sequence make a network of close contacts to form a stable tertiary structure. In contrast, ankyrin repeats have regular linear
mNotch; EP domain 1
mNotch; EP domain 2
mNotch; EP domain 3
NSu42c
sy56
3
4
5
6
7
A1034T G1043E G1057E
R1029W
D2098A, I 2099A, V2100A V2100A, R2101A, L2102A L2103A, D2104A, E2105A V2062A
G1985A, T1987A, L1989A A1992E, A1993F
Residue #
Sev-ANK
CDCN1T
RAMANK
CDC23
⌬2105
1101
12
13
14
15
16
17
2102–2223
2078stop, rNotch1 2098stop, rNotch1 2125stop, Xotch 2105–2114
2108stop, dNotch
2127stop, dNotch
Beatus et al., 2001
Jeffries et al., 2002
Freyer et al., 2002
Kurooka et al., 1998
Shawber et al., 1996
Matsuno et al., 1997b
Lyman and Young, 1993
Kodoyianni et al., 1992 Kodoyianni et al., 1992 Kodoyianni et al., 1992
Kodoyianni et al., 1992
Diederich et al., 1994
Oswald et al., 2001
Oswald et al., 2001
Oswald et al., 2001
Kopan et al., 1994
Kopan et al., 1994
Reference ⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫹
⫹/⫺
⫺
⫺
⫹
⫹
⫺
⫺
EMSA
ND
ND
ND
⫺
⫺
MYO
loss-of-function
loss-of-function
Genetic Definition
hypomorphic; a weak Notch function in Su(H)-mediated decisions temperature sensitive (TS), loss-of-function at restrictive (high) temperature TS TS TS
IP
HES
⫺
⫹
⫺
⌬7
⫺
⌬7
⫺
⫺
⌬7
⌬7
ND
⌬7
ND
⫹
ND
⫺
⫺
ND
⫹
⫹
ND
⫺
ND
ND
ND
ND
ND
⫹
ND
No binding to MAM1
Overexpression has a phenotype
Loss-of-function. Interacts with Wg and possibly Deltex. Affects nerve progenitors (Brennan et al., 1997)
Functional Properties
4 4 4
4
5
7
7
7
4
4
ANK
Functional Properties
Would not affect the ANK domain stability Predicted to destabilize ANK domain Predicted to destabilize ANK domain
same as above
same as above
Would reduce overall stability due to loss of 7th ankyrin repeat (Zweifel and Barrick, 2001a) same as above
Predicted Effect on Stability
none (Zweifel et al., 2003)
Predicted to destabilize 7th ankyrin repeat (see text)
loss of secondary structure, reduced stability (Zweifel., et al., 2003)
reduced stability (Zweifel., et al., 2003)
Impact on Stability
Abbreviations used in the table: HES (Hes), Luc activation assay; IP, any type of physical association assay with CSL proteins, immunoprecipitations, or yeast two-hybrid assays; EMSA, electrophoresis mobility shift assay; Myo, inhibition of myogenesis; ND, not determined. Information in the table was compiled from additional publications, not all cited in this review.
N g
11
60 11
Deletion in ANK Domains Ending at
glp-1; bn18 glp-1; q224 glp-1; q231
mNotch; M2
2
8 9 10
mNotch; M1
1
Mutation; construct
Point Mutations in ANK Domains
Table 1. Selected Mutations and Deletions in Notch1 Proteins from Different Organisms and Their Predicted Effect on Stability
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topology where residues closely positioned in the primary sequence form contacts between neighboring repeats and lack long-range network interactions. Surprisingly, in spite of such modular architecture, ankyrin repeats do not fold independently: previous proteolysis and mutagenesis studies suggest that at least two repeats are necessary to form a stably folded structure, indicating that some long-range communication between repeats must exist (Michaely and Bennett, 1993). However, different ankyrin repeats contribute unequally to the overall stability of the ANK domain. To address the stability and folding properties of the Notch ANK domain, the effect of substituting a single conserved Ala to Gly in each individual repeat (Bradley and Barrick, 2002) and the effect of deleting repeats six and seven was evaluated (Zweifel and Barrick, 2001a, 2001b). The wild-type protein sequence and sequence constructs containing a single mutation in either of repeats two through five unfolded as a single unit, while the mutation in repeat six reduced the degree of cooperativity during unfolding. Based on their thermodynamic properties, repeats six and seven appear to comprise an independent subdomain. At the same time, deletion of repeat seven caused unfolding of repeat six and a 50% decrease in the overall stability of the ANK domain (Bradley and Barrick, 2002). The observed long-range communication between repeats two to seven may be explained by the presence of an elongated hydrophobic core at the junctions of  hairpin linkers with ␣ helices, which extended through all repeats as seen in the crystal structure. Overall, two significant conclusions emerged from biophysical studies: first, long-range, asymmetric communication exists between N- and C-terminal repeats, and second, the seventh ankyrin repeat is important to the folding and stability of the entire ANK domain. What is the functional significance of this data to the existence of the EP or RE/AC domain defined by mutating or deleting this repeat?
The EP Domain: p300 Binding or ANK Domain Stability? The functional importance of the region around the seventh ankyrin repeat for the transcription activation was demonstrated through numerous mutagenesis studies, but the proposed mechanism of its activity as an independent HAT/cofactor interaction domain (EP-RE/AC) needs to be revisited in light of the structural and biophysical studies presented above. As introduced earlier, in vitro reconstruction of the Notch transcription complex as well as previous genetic analyses (summarized in Mumm and Kopan, 2000) arrived at the conclusion that the first step toward a successful assembly of the transcription-activating complex is a Notch/CSL interaction. This creates an interface to permit a second, non-DNA binding transcriptional coactivator (MAM) to bind (Doyle et al., 2000; Nam et al., 2003; Petcherski and Kimble, 2000; Wallberg et al., 2002; Wu et al., 2000). NICD can coimmunoprecipitate with p300 (Oswald et al., 2001; Wallberg et al., 2002), but in complex with RBPjk, it is unable to recruit the HAT p300 to DNA in cell free systems (Fryer et al., 2002; Wallberg et al.,
Figure 3. The Contribution of Specific Amino Acids to the Hydrophobic Core between Ank Repeats Six and Seven The chain trace of Drosophila Notch ankyrin repeats six (yellow), seven (cyan), and  turn in between (gray) is shown in “worm” representation. Residues I232, L235 (shown in ball-and-stick representation in blue), and L236 (shown in red) corresponds to mNotch residues I2097, L2100, and L2103, mutated in mNotch (Oswald et al., 2001) (see Table 1, rows three to five). Residues L188 and L204 (shown in orange) together with L236 form the hydrophobic core important for the overall stability of the ANK domain.
2002). In contrast, p300 interacts strongly with MAM even without the benefit of having Notch present (Wallberg et al., 2002). These studies collectively suggest that addition of MAM is essential for the Notch/RBPjk complex to recruit p300 to DNA, further substantiated by the nuclear accumulation and phosphorylation of p300 upon addition of MAM (Fryer et al., 2002; Oswald et al., 2001; Wallberg et al., 2002). Numerous deletion studies found that the region containing the seventh ankyrin repeat was critical for Notch to activate transcription while the full TAD was required for maximal transcription activation (Table 1). Fryer and colleagues studied CDC23, a Notch protein with a deletion C-terminal to the critical EP domain/seventh ankyrin repeat (number 15 in Figure 2, Table 1), and found it to be the minimal construct able to activate transcription. In a similar study, deletion of the seventh ankyrin repeat (⌬2105; Jeffries et al., 2002) was sufficient to abrogate the ability of Notch to activate transcription; this protein could not be rescued by the addition of MAM1. While purified Notch proteins can weakly interact with p300, perhaps through more distal regions within the TAD domain (Kurooka and Honjo, 2000; Wallberg et al., 2002), the EP domain was suggested to play a key role in mediating the interaction between p300 and the NICD (Oswald et al., 2001). Of the residues mutated in this region, only the substitution of LDE to AAA abolished Notch-mediated transcriptional activation (Oswald et al., 2001), interpreted by the authors to suggest a role for these amino
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Figure 4. Diagram of the Hypothetical Mechanism by which Notch Converts the CSL Corepressor Complex to a Transcriptional Coactivator Complex Once in the nucleus, NICD displaces the corepressor complex in a manner that is only partially understood. This displacement and subsequent conversion to an activator complex occurs by direct protein-protein interactions between the NICD, SKIP, and CSL, which lead to SMRT/ HDAC dissociation. The latter is sufficient to activate transcription on some targets (i.e, the single-minded gene). For most other targets and for in vitro transcription, the transcriptional activator assembles in steps. First the Notch/CSL interface recruits Mastermind, then the triprotein complex recruits histone acetylases (p300, others) and RNA polymerase II, resulting in activation of of Hes/E(spl) and other downstream targets of Notch. NICD ubiquitination and subsequent degradation by the proteasome is thought to terminate the activation phase, permitting reassembly of the repressor complex. Abbreviations: SMRT, silencing mediator of retinoid and thyroid hormone receptors; SKIP, ski-related protein; HDAC, histone deacetlyase; HAT, histone acetylase; other abbreviations are provided in the text (see text for further details).
acids in mediating direct interaction with p300. The crystal structure reveals that Asp or Glu in the LDE motif are exposed on opposite surfaces of ankyrin repeat seven and could potentially contribute to direct contacts with p300, as suggested by Oswald and colleagues (Oswald et al., 2001). However, Leu 2103 in the LDE motif (Figure 3; L236 in the crystal) is buried within the structure and is the only invariant hydrophobic residue in this helix that participates in the maintenance of the common, elongated hydrophobic core formed by side chains within ankyrin repeat six, the  hairpin linker, and ankyrin repeat seven (Figure 2, mutation 5; Figure 3). Therefore, removal of this hydrophobic side chain by an Ala substitution could decrease considerably the stability of the entire ANK domain (Figures 2 and 3). Compounding the effect of this mutation is the loss of a double salt bridge present between the side chain of Asp in LDE and Arg2085 located in the first ␣ helix in ankyrin repeat seven (R221 in the crystal), which could contribute to the proper folding of this repeat (Figure 2). Destabilization of the ANK domain will most likely affect the direct interaction with MAM; subsequently, if MAM cannot be properly recruited to the NICD/RBPjk complex, the interface required for the recruitment of p300 may not form properly. Support for this comes from analysis of the structural properties of known mutations in the ANK domain (Zweifel et al., 2003; Table 1,
Figure 2). Partial loss-of-function mutations are mapped to surface-exposed residues and thus preserve the overall structure and stability of the ANK domain (all glp-1 mutations in C. elegans, Nsu42c in Drosophila Notch). In contrast, lethal loss-of-function mutations either disrupt tertiary structure (M2) or compromise the stability of the ANK domain (M1). Interestingly, like the LDE mutation, M1 targeted a conserved leucine (L1989A) that makes an analogous contribution to the hydrophobic core near ankyrin repeat four. It is important to mention that residues mutated in M1 and M2 in the fourth ankyrin repeat preserve the integrity of the EP domain. Both mutant NICD proteins bind to RBPjk (Jarriault et al., 1995; and R.K., unpublished data) but are unable to activate transcription (Jarriault et al., 1995; Kato et al., 1997; Kurooka et al., 1998); we interpret that to suggest that the EP domain could not recruit p300 to RBPjk when the ANK domain is destabilized. When MAM is in excess, it can form a trimeric protein complex with RBP and an intact ANK domain missing RAM (Jeffries et al., 2002). Given that addition of an intact ANK domain “rescues” the M1 mutants (Kurooka et al., 1998), the most likely interpretation for this group of experimental observations is that MAM can bridge between the RAM-less NICD and RBPjk, forming an interface for proper binding of p300. If the stability of the ANK domain is compromised by mutations or deletion of
Review 625
the seventh repeat that interface fails to form due to global effects on the ANK domain. Finally, several prooncogenic mutations in another ankyrin repeat protein— the tumor suppressor p16INK4A (Tang et al., 1999; Tevelev et al., 1996)—impact amino acids that, like Leu 2103 in Drosophila Notch, are not involved in protein interactions but rather are buried inside the structure and affect the stability of other ankyrin repeats (Tang et al., 2003; Zhang and Peng, 2002). In summary, the demonstration that the ANK domain includes a seventh repeat solidifies a large number of experimental results obtained by various methods into a more coherent view of the relationship between the Notch structure and its function. Previously, the failure to activate transcription by proteins lacking the EP/RE/ AC domain had been attributed to the loss of direct interaction of this domain with HATs or other cofactors; alternatively, in light of the literature reviewed here, it is more likely that these studies mapped the biological importance of the entire ANK domain due to the peculiarities of its folding and the resultant failure to properly recruit MAM (Figure 4). Interestingly, it has been recently noted that phosphorylation near the seventh repeat (Espinosa et al., 2003; Foltz et al., 2002; Ingles-Esteve et al., 2001) may target NICD to degradation and thus reduce its activity; given the newest structural information on what regions constitute the ANK domain, it would be interesting to test if the observed change in NICD half-life is caused by alterations in the seventh ankyrin repeat and thus in overall structural stability of the Notch cleavage product. We move forward through integration of newfound knowledge into our working models. Deletion constructs, similar to the ones highlighted here, are widely used in biological experiments designed to decipher the signaling properties and biological roles of Notch proteins; we need to reexamine those constructs that removed amino acids within the (correctly defined) ANK domain and refine the conclusions drawn from using them. More insight will undoubtedly be gained as additional structural information emerges describing Notch-pathway proteins and their complexes. Finally, a universally applicable lesson is that integration of “hard” structural and biophysical data with the biochemical and genetic observations will be critical for comprehensive understanding of all protein functions.
domains is critical for regulation of HES promoter activity. Mech. Dev. 104, 3–20.
Acknowledgments
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.
We are indebted to Dr. Gabriel Waksman for his support and encouragement during the early phases of this work and to Dr. Malu Tansey for critical reading of the manuscript; to both we are grateful for many thoughtful discussions. R.K. is supported by NIH grant GM55479, O.Y.L. by a fellowship from the Keck foundation, S.V.K. by the E.A. Doisy Trust. References Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770–776. Ball, D.W., and Leach, S.D. (2003). Notch in malignancy. Cancer Treat. Res. 115, 95–121. Beatus, P., Lundkvist, J., Oberg, C., Pedersen, K., and Lendahl, U. (2001). The origin of the ankyrin repeat region in Notch intracellular
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Jeffries, S., Robbins, D.J., and Capobianco, A.J. (2002). Characterization of a high-molecular-weight notch complex in the nucleus of Notch(ic)-transformed RKE cells and in a human T-cell leukemia cell line. Mol. Cell. Biol. 22, 3927–3941. Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M., and Kadesch, T. (1998). A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269–2277. Kato, H., Taniguchi, Y., Kurooka, H., Minoguchi, S., Sakai, T., Nomura-Okazaki, S., Tamura, K., and Honjo, T. (1997). Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. Development 124, 4133–4141. 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.
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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.
Blacklow, S.C. (2003). Nuclear magnetic resonance structure of a prototype lin12-notch repeat module from human notch1. Biochemistry 42, 7061–7067.
Kurooka, K., and Honjo, T. (2000). Functional interaction between the mouse Notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J. Biol. Chem. 275, 17211–17220.
Wallberg, A.E., Pedersen, K., Lendahl, U., and Roeder, R.G. (2002). p300 and PCA act cooperatively to mediate transcriptional activation from chromatin templates by Notch intracellular domains in vitro. Mol. Cell. Biol. 22, 7812–7819.
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.
Wettstein, D.A., Turner, D.L., and Kintner, C. (1997). The Xenopus homolog of Drosophila suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124, 693–702.
Lyman, D., and Young, M.W. (1993). Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc. Natl. Acad. Sci. USA 90, 10395–10399.
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.
Maillard, I., and Pear, W.S. (2003). Notch and cancer: best to avoid the ups and downs. Cancer Cell 3, 203–205. 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. Michaely, P., and Bennett, V. (1993). The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats. J. Biol. Chem. 268, 22703–22709. Mumm, J.S., and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228, 151–165. Nam, Y., Aster, J.C., and Blacklow, S.C. (2002). Notch signaling as a therapeutic target. Curr. Opin. Chem. Biol. 6, 501–509. Nam, Y., Weng, A.P., Aster, J.C., and Blacklow, S.C. (2003). Structural requirements for assembly of the CSL/intracellular notch1/ mastermind-like 1 transcriptional activation complex. J. Biol. Chem. 278, 21232–21239. Nicolas, M., Wolfer, A., Raj, K., Kummer, J.A., Mill, P., Van Noort, M., Hui, C.C., Clevers, H., Dotto, G.P., and Radtke, F. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nat. Genet. 33, 416–421. Oswald, F., Tauber, B., Dobner, T., Bourteele, S., Kostezka, U., Adler, G., Liptay, S., and Schmid, R.M. (2001). p300 acts as a transcriptional coactivator for mammalian Notch-1. Mol. Cell. Biol. 21, 7761–7774. Petcherski, A.G., and Kimble, J. (2000). LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway. Nature 405, 364–368. Rao, Z., Handford, P., Mayhew, M., Knott, V., Brownlee, G.G., and Stuart, D. (1995). The structure of a Ca(2⫹)-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82, 131–141. 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. Shawber, C., Nofziger, D., Hsieh, J.J., Lindsell, C., Bogler, O., Hayward, D., and Weinmaster, G. (1996). Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122, 3765–3773. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., and Honjo, T. (1995). Physical interaction between a novel domain of the receptor Notch and the transcription factor Rbp-JKappa/Su(H). Curr. Biol. 5, 1416–1423. 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. Tang, K.S., Fersht, A.R., and Itzhaki, L.S. (2003). Sequential unfolding of ankyrin repeats in tumor suppressor p16. Structure 11, 67–73. Tani, S., Kurooka, H., Aoki, T., Hashimoto, N., and Honjo, T. (2001). The N- and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate transcription. Nucleic Acids Res. 29, 1373–1380. 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. Vardar, D., North, C.L., Sanchez-Irizarry, C., Aster, J.C., and
Wu, L., Aster, J.C., Blacklow, S.C., Lake, R., Artavanis-Tsakonas, S., and Griffin, J.D. (2000). MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26, 484–489. Zhang, B., and Peng, Z.Y. (2002). Structural consequences of tumorderived mutations in p16INK4a probed by limited proteolysis. Biochemistry 41, 6293–6302. Zhou, S., and Hayward, S.D. (2001). Nuclear localization of CBF1 is regulated by interactions with the SMRT corepressor complex. Mol. Cell. Biol. 21, 6222–6232. Zhou, S., Fujimuro, M., Hsieh, J.J., 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. Zweifel, M.E., and Barrick, D. (2001b). Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding. Biochemistry 40, 14357–14367. Zweifel, M.E., Leahy, D.J., Hughson, F.M., and Barrick, D. (2003). Structure and stability of the ankyrin domain of the Drosophila Notch receptor. Protein Sci. 12, 2622–2632.
Anchoring Notch Genetics and Review Biochemistry: Structural Analysis of the Ankyrin Domain Sheds Light on Existing Data Olga Y. Lubman,1 Sergey V. Korolev,2 and Raphael Kopan1,* 1 Department of Molecular Biology and Pharmacology and the Department of Medicine Washington University School of Medicine 2 Edward A. Doisy Department of Biochemistry and Molecular Biology Saint Louis University School of Medicine Saint Louis, Missouri 63104
Notch signaling is important in development and in human disease. Notch receptors regulate transcription through direct interactions with several proteins at the promoter regions of target genes. To understand the mechanism of Notch signaling, numerous deletion and mutagenesis studies have been carried out to identify functional domains in Notch, but domain definition and their role during the assembly of the transcriptionally active complex remains controversial. Recently reported biophysical and structural studies of the Notch ANK domain permit us to reevaluate the existing domain assignments and their predicted functional role, thereby providing further insight into the mechanism of Notch signaling.
Notch is an essential part of an ubiquitous signaling pathway involved in a variety of cell fate decisions in metazoans (Artavanis-Tsakonas et al., 1999; Bray, 1998). Mutations in Notch pathway genes cause several human disorders (Gridley, 2003) and can either promote (Ball and Leach, 2003; Ellisen et al., 1991; Maillard and Pear, 2003; Nam et al., 2002) or suppress (Nicolas et al., 2003) cancer. Since the discovery of the Notch locus by Dexter in 1914, studies of the Notch signaling pathway progressed for eight decades without the scrutiny of structural biology. Over the last decade and most recently in a publication describing the crystal structure of the Notch ankyrin repeats (Zweifel et al., 2003), structural biologists joined their distinctive voice to the chorus of Notch investigators. In this review, we will integrate the insights gained from structural and biophysical studies of the Drosophila Notch ankyrin repeats with those gained by genetic and biochemical experiments in many organisms. It is our view that several domain assignments made previously on the basis of biochemical and genetic experiments will require redefinition in light of this critical new information. The Notch protein is a large (300 kDa) single-pass type I transmembrane receptor activated by a regulated intramembrane proteolysis (RIP; Brown et al., 2000). Defined structural domains in the extracellular domain of
*Correspondence: [email protected]
Notch include EGF (Figure 1, yellow; Rao et al., 1995) and LNR (Lin-Notch repeats, orange; Vardar et al., 2003) repeats. Signaling through Notch is initiated by binding of surface-anchored ligands to EGF-like repeats 11 and 12 (shaded) in the extracellular portion of the receptor. The binding of ligand induces proteolysis that sheds the extracellular portion of the Notch receptor. This is followed by a second cleavage hydrolyzing a peptide bond within the transmembrane domain; as a result, the Notch intracellular domain (NICD) dissociates from the membrane and enters the nucleus (for review see Fortini, 2002; Mumm and Kopan, 2000). Instead of possessing an enzymatic activity, NICD orchestrates changes in DNA-bound protein assemblies in the nucleus, centered around the DNA binding protein CSL [CBP or RBPjk in vertebrates, Su(H), Drosophila, Lag-1 in C. elegans; we will mention specific proteins by name when describing data obtained with that protein]. In the absence of Notch, RBPjk interacts with the proteins SKIP and SMRT to recruit one of several transcriptional corepressor complexes (Zhou et al., 2000; Zhou and Hayward, 2001). Therefore, successful signaling via Notch receptors depends on the ability of NICD to displace the corepressor complex associated with RBPjk and convert it to a transcriptionally active complex. Two domains in NICD involved in its interaction with CSL were defined by several nonbiophysical methods. The first was discovered in a yeast two-hybrid assay; it is called the RAM (RBPjk-associated molecule) domain (Tamura et al., 1995). While some critical amino acids for RAM domain/RBPjk interactions were identified by mutagenesis, the exact boundaries of this domain are poorly defined. The RAM domain spans the region from just beyond the Arg/Lys cluster (serving as stop translocation signal at the cytosolic side of the transmembrane domain) to the first ankyrin repeat (Figure 1, magenta). Defining the exact boundaries of the RAM domain is further complicated by the fact that this region appears to be unstructured as evidenced by CD spectroscopy measurements (Nam et al., 2003). Sequence similarity recognized the second domain as a cluster of ankyrin repeats (ANK domain), a highly conserved motif (Figure 1, blue) responsible for mediating protein interactions (Bork, 1993; Groves and Barford, 1999; Sedgwick and Smerdon, 1999; Wharton et al., 1985). Genetic experiments demonstrated that the ANK domain is essential for all known Notch functions (Artavanis-Tsakonas et al., 1999; Greenwald, 1994). NICD interacts strongly with CSL via the RAM domain with the ANK domain contributing to this interaction (Jarriault et al., 1995; Kato et al., 1997; Kodoyianni et al., 1992; Matsuno et al., 1997; Tani et al., 2001). Once the NICD/CSL complex is formed, the protein Mastermind/SEL-8/LAG-3 (MAM) binds to this complex (Doyle et al., 2000; Nam et al., 2003; Petcherski and Kimble, 2000; Wallberg et al., 2002; Wu et al., 2000). Histone acetyltransferases (HAT) are recruited next (Fryer et al., 2002; Kao et al., 1998; Oswald et al., 2001; Wallberg et al., 2002) to assemble the active transcription complex. The domain thought to mediate direct
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Figure 1. Domain Organization of Notch Receptors This diagram uses mouse Notch1 as a prototype. Proteolytic cleavage by furin at site 1 (S1) produces two subunits, which remain noncovalently associated at the cell surface. EGF-like modules 11 and 12 participate in ligand binding (shaded, EGF repeats in yellow). Two consecutive proteolytic cleavages induced upon ligand activation release the intracellular domain of Notch (NICD). NICD consists of the RAM domain (magenta), NLS (nuclear localization signal), ankyrin repeats (blue), TAD (transactivation domain, black), and STR-Ser/Thr-rich domain regulating cytokine response. PEST (proline, glutamate, serine, threonine-rich) sequence and OPA (glutamine-rich sequence) motifs are located C-terminal to the ankyrin repeats; the former regulates protein turnover while the latter contains an “acidic blob” that is thought to enhance transcriptional activation by Notch proteins. (A) EP domain, which maps just C-terminal to the sixth ankyrin repeat, was defined as the region mediating direct interaction with the HAT (histone acetylases) protein p300 (red). (B) Crystal structure of the Drosophila ANK domain confirmed the presence of the seventh repeat that overlaps with the EP domain. The first “ankyrin repeat” does not adopt a regular ankyrin fold and is shaded; the overall number of ankyrin repeats that adopt a canonical ankyrin fold is six. (C) Ribbon representation of the structure of Drosophila ANK domain (Zweifel et al., 2003). The repeats are numbered consecutively.
interaction between Notch and HAT proteins PCAF and GCN5 was mapped to a region C-terminal to the ANK domain and designated the transactivation domain (TAD; Kurooka and Honjo, 2000). Studies using purified proteins demonstrate that the presence of MAM is essential for the recruitment of the HAT protein p300 (Fryer et al., 2002; Wallberg et al., 2002); others propose that direct interaction between Notch and p300 contributes to the recruitment of p300 to the complex (Kurooka and Honjo, 2000; Oswald et al., 2001; Wallberg et al., 2002). Further dissection of TAD using deletion mutagenesis identified the smaller “EP” domain (included in the RE/ AC domain [Beatus et al., 2001] and at the N-terminal boundary of the TAD domain) as critical for transcription activation, potentially through interaction with p300 (Oswald et al., 2001). The question, which needs to be examined in light of the structural studies, is this: do the amino acids included in the EP domain constitute an additional independent subdomain of Notch, and if so, what are its boundaries and its functional significance? Based on the crystal structure of the Drosophila Notch ANK domain and biophysical experiments, the deletion experiments aimed at understanding the function of this region in Notch may have instead demonstrated (again) the importance of the ANK domain to Notch function.
Newly Defined Boundaries of the Notch ANK Domain The placement of the ANK domain in most published Notch sequences was based on homology with available consensus sequences (Bork, 1993; Breeden and Nasmyth, 1987; Kodoyianni et al., 1992; Wharton et al., 1985). Although in their experimental design most authors considered the intracellular portion of Notch receptors to contain only six ankyrin repeats, some recognized the presence of the seventh ankyrin repeat (Bork, 1993; Wettstein et al., 1997). Evidence supporting the existence of a seventh repeat in the archetypical Drosophila Notch were provided by urea-induced unfolding experiments and other biophysical measurements (Nam et al., 2003; Zweifel and Barrick, 2001a, 2001b) and was unequivocally confirmed by the 2.0 A˚ crystal structure of the ANK domain obtained by Barrick and colleagues (Zweifel et al., 2003). The crystal structure further revised the placement of the repeats because it demonstrated that the first (most N-terminal) ankyrin repeat does not adopt a regular ankyrin fold, although we can not completely rule out that CSL binding might induce substantial conformational changes in this region allowing it to adopt the ankyrin fold conformation. Regardless of whether the Notch intracellular domain has six or seven properly folded ankyrin repeats, the crystal structure has
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Figure 2. Natural and Synthetic Mutations in the Ankyrin Domain of Metazoan Notch-1 Proteins and Their Location Relative to the Crystal Structure Structure-based sequence alignment of the ANK domain of Notch-1 orthologs from six different species: D. melanogaster (Dm; AC# AH005262), M. musculus (Mm; AC# NP032740), H. sapiens (Hs; AC# P46531), X. Laevis (Xl; AC# XP342393), R. norvegicus (Rn; AC# NP077334), and glp-1 from C. elegans (Ce; AC# A32901). The alignment was generated using programs CLUSTALV and ESPcript (ExPASy Molecular Biology Server; www.expasy.org). Identical residues are shown in white on black background and homologous residues are shown on gray background. Note that reduced homology is the consequence of including glp-1 in this alignment. Helices of each repeat from the structure of Drosophila ANK domain (Zweifel et al., 2003) are represented by cylinders above the primary sequences. The boundaries of the TAD and EP domains are shown as blue and orange bars, respectively. The mutations are numbered according to their order of appearance in Table 1 in which their molecular nature, behavior in assays, and impact on structure (when known) are described. Mutations and truncations that completely disrupt Notch functions are highlighted in red, while those with milder phenotype (hypomorphic or temperature sensitive) are highlighted in cyan. The Ala substitution of DI(V)_(V)RL_LDE residues is shown in mutation 3–5; note that Val2099 is mutated in both constructs 3 and 4. Many Notch truncations within this ankyrin repeat were published; colored lines indicate the C-terminal boundary of few selected truncated-Notch constructs used by many investigators in Notch field. The citations in Table 1 credit the labs that created or described the truncation.
redefined the C-terminal boundary of the ANK domain by including an additional repeat (Figure 1). What is the relevance of extending the ANK domain for interpretation of cell-based functional analyses in which part of this region was deleted to define the roles of the EP, RE/AC, and TAD domains? To answer this question, we need to first understand what ankyrin repeats are and to examine the contribution of the seventh ankyrin repeat to the stability and folding of the entire ANK domain. Cooperative Unfolding of Notch Ankyrin Repeats The ankyrin repeat is an array of ␣-helical motifs positioned in tandem that is found in a variety of proteins responsible for mediating protein-protein interactions necessary for transcriptional regulation, cell-cycle control, cytoskeletal organization, and cell differentiation
(Breeden and Nasmyth, 1987). Despite considerable sequence variability, ankyrin repeat-containing proteins show very regular and conserved secondary and tertiary structure. Each repeat consists of approximately 33 residues forming two antiparallel ␣ helices connected by a short loop (Figure 2). The adjacent repeats are packed via hydrophobic interactions and are connected by a  hairpin, extended perpendicular to the helical axes to form an L shape structure (Figure 1; Sedgwick and Smerdon, 1999). The elongated tertiary structure of ankyrin repeatcontaining proteins differs significantly from that seen in typical globular proteins, where multiple residues distant from each other in the primary sequence make a network of close contacts to form a stable tertiary structure. In contrast, ankyrin repeats have regular linear
mNotch; EP domain 1
mNotch; EP domain 2
mNotch; EP domain 3
NSu42c
sy56
3
4
5
6
7
A1034T G1043E G1057E
R1029W
D2098A, I 2099A, V2100A V2100A, R2101A, L2102A L2103A, D2104A, E2105A V2062A
G1985A, T1987A, L1989A A1992E, A1993F
Residue #
Sev-ANK
CDCN1T
RAMANK
CDC23
⌬2105
1101
12
13
14
15
16
17
2102–2223
2078stop, rNotch1 2098stop, rNotch1 2125stop, Xotch 2105–2114
2108stop, dNotch
2127stop, dNotch
Beatus et al., 2001
Jeffries et al., 2002
Freyer et al., 2002
Kurooka et al., 1998
Shawber et al., 1996
Matsuno et al., 1997b
Lyman and Young, 1993
Kodoyianni et al., 1992 Kodoyianni et al., 1992 Kodoyianni et al., 1992
Kodoyianni et al., 1992
Diederich et al., 1994
Oswald et al., 2001
Oswald et al., 2001
Oswald et al., 2001
Kopan et al., 1994
Kopan et al., 1994
Reference ⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫹
⫹/⫺
⫺
⫺
⫹
⫹
⫺
⫺
EMSA
ND
ND
ND
⫺
⫺
MYO
loss-of-function
loss-of-function
Genetic Definition
hypomorphic; a weak Notch function in Su(H)-mediated decisions temperature sensitive (TS), loss-of-function at restrictive (high) temperature TS TS TS
IP
HES
⫺
⫹
⫺
⌬7
⫺
⌬7
⫺
⫺
⌬7
⌬7
ND
⌬7
ND
⫹
ND
⫺
⫺
ND
⫹
⫹
ND
⫺
ND
ND
ND
ND
ND
⫹
ND
No binding to MAM1
Overexpression has a phenotype
Loss-of-function. Interacts with Wg and possibly Deltex. Affects nerve progenitors (Brennan et al., 1997)
Functional Properties
4 4 4
4
5
7
7
7
4
4
ANK
Functional Properties
Would not affect the ANK domain stability Predicted to destabilize ANK domain Predicted to destabilize ANK domain
same as above
same as above
Would reduce overall stability due to loss of 7th ankyrin repeat (Zweifel and Barrick, 2001a) same as above
Predicted Effect on Stability
none (Zweifel et al., 2003)
Predicted to destabilize 7th ankyrin repeat (see text)
loss of secondary structure, reduced stability (Zweifel., et al., 2003)
reduced stability (Zweifel., et al., 2003)
Impact on Stability
Abbreviations used in the table: HES (Hes), Luc activation assay; IP, any type of physical association assay with CSL proteins, immunoprecipitations, or yeast two-hybrid assays; EMSA, electrophoresis mobility shift assay; Myo, inhibition of myogenesis; ND, not determined. Information in the table was compiled from additional publications, not all cited in this review.
N g
11
60 11
Deletion in ANK Domains Ending at
glp-1; bn18 glp-1; q224 glp-1; q231
mNotch; M2
2
8 9 10
mNotch; M1
1
Mutation; construct
Point Mutations in ANK Domains
Table 1. Selected Mutations and Deletions in Notch1 Proteins from Different Organisms and Their Predicted Effect on Stability
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Review 623
topology where residues closely positioned in the primary sequence form contacts between neighboring repeats and lack long-range network interactions. Surprisingly, in spite of such modular architecture, ankyrin repeats do not fold independently: previous proteolysis and mutagenesis studies suggest that at least two repeats are necessary to form a stably folded structure, indicating that some long-range communication between repeats must exist (Michaely and Bennett, 1993). However, different ankyrin repeats contribute unequally to the overall stability of the ANK domain. To address the stability and folding properties of the Notch ANK domain, the effect of substituting a single conserved Ala to Gly in each individual repeat (Bradley and Barrick, 2002) and the effect of deleting repeats six and seven was evaluated (Zweifel and Barrick, 2001a, 2001b). The wild-type protein sequence and sequence constructs containing a single mutation in either of repeats two through five unfolded as a single unit, while the mutation in repeat six reduced the degree of cooperativity during unfolding. Based on their thermodynamic properties, repeats six and seven appear to comprise an independent subdomain. At the same time, deletion of repeat seven caused unfolding of repeat six and a 50% decrease in the overall stability of the ANK domain (Bradley and Barrick, 2002). The observed long-range communication between repeats two to seven may be explained by the presence of an elongated hydrophobic core at the junctions of  hairpin linkers with ␣ helices, which extended through all repeats as seen in the crystal structure. Overall, two significant conclusions emerged from biophysical studies: first, long-range, asymmetric communication exists between N- and C-terminal repeats, and second, the seventh ankyrin repeat is important to the folding and stability of the entire ANK domain. What is the functional significance of this data to the existence of the EP or RE/AC domain defined by mutating or deleting this repeat?
The EP Domain: p300 Binding or ANK Domain Stability? The functional importance of the region around the seventh ankyrin repeat for the transcription activation was demonstrated through numerous mutagenesis studies, but the proposed mechanism of its activity as an independent HAT/cofactor interaction domain (EP-RE/AC) needs to be revisited in light of the structural and biophysical studies presented above. As introduced earlier, in vitro reconstruction of the Notch transcription complex as well as previous genetic analyses (summarized in Mumm and Kopan, 2000) arrived at the conclusion that the first step toward a successful assembly of the transcription-activating complex is a Notch/CSL interaction. This creates an interface to permit a second, non-DNA binding transcriptional coactivator (MAM) to bind (Doyle et al., 2000; Nam et al., 2003; Petcherski and Kimble, 2000; Wallberg et al., 2002; Wu et al., 2000). NICD can coimmunoprecipitate with p300 (Oswald et al., 2001; Wallberg et al., 2002), but in complex with RBPjk, it is unable to recruit the HAT p300 to DNA in cell free systems (Fryer et al., 2002; Wallberg et al.,
Figure 3. The Contribution of Specific Amino Acids to the Hydrophobic Core between Ank Repeats Six and Seven The chain trace of Drosophila Notch ankyrin repeats six (yellow), seven (cyan), and  turn in between (gray) is shown in “worm” representation. Residues I232, L235 (shown in ball-and-stick representation in blue), and L236 (shown in red) corresponds to mNotch residues I2097, L2100, and L2103, mutated in mNotch (Oswald et al., 2001) (see Table 1, rows three to five). Residues L188 and L204 (shown in orange) together with L236 form the hydrophobic core important for the overall stability of the ANK domain.
2002). In contrast, p300 interacts strongly with MAM even without the benefit of having Notch present (Wallberg et al., 2002). These studies collectively suggest that addition of MAM is essential for the Notch/RBPjk complex to recruit p300 to DNA, further substantiated by the nuclear accumulation and phosphorylation of p300 upon addition of MAM (Fryer et al., 2002; Oswald et al., 2001; Wallberg et al., 2002). Numerous deletion studies found that the region containing the seventh ankyrin repeat was critical for Notch to activate transcription while the full TAD was required for maximal transcription activation (Table 1). Fryer and colleagues studied CDC23, a Notch protein with a deletion C-terminal to the critical EP domain/seventh ankyrin repeat (number 15 in Figure 2, Table 1), and found it to be the minimal construct able to activate transcription. In a similar study, deletion of the seventh ankyrin repeat (⌬2105; Jeffries et al., 2002) was sufficient to abrogate the ability of Notch to activate transcription; this protein could not be rescued by the addition of MAM1. While purified Notch proteins can weakly interact with p300, perhaps through more distal regions within the TAD domain (Kurooka and Honjo, 2000; Wallberg et al., 2002), the EP domain was suggested to play a key role in mediating the interaction between p300 and the NICD (Oswald et al., 2001). Of the residues mutated in this region, only the substitution of LDE to AAA abolished Notch-mediated transcriptional activation (Oswald et al., 2001), interpreted by the authors to suggest a role for these amino
Molecular Cell 624
Figure 4. Diagram of the Hypothetical Mechanism by which Notch Converts the CSL Corepressor Complex to a Transcriptional Coactivator Complex Once in the nucleus, NICD displaces the corepressor complex in a manner that is only partially understood. This displacement and subsequent conversion to an activator complex occurs by direct protein-protein interactions between the NICD, SKIP, and CSL, which lead to SMRT/ HDAC dissociation. The latter is sufficient to activate transcription on some targets (i.e, the single-minded gene). For most other targets and for in vitro transcription, the transcriptional activator assembles in steps. First the Notch/CSL interface recruits Mastermind, then the triprotein complex recruits histone acetylases (p300, others) and RNA polymerase II, resulting in activation of of Hes/E(spl) and other downstream targets of Notch. NICD ubiquitination and subsequent degradation by the proteasome is thought to terminate the activation phase, permitting reassembly of the repressor complex. Abbreviations: SMRT, silencing mediator of retinoid and thyroid hormone receptors; SKIP, ski-related protein; HDAC, histone deacetlyase; HAT, histone acetylase; other abbreviations are provided in the text (see text for further details).
acids in mediating direct interaction with p300. The crystal structure reveals that Asp or Glu in the LDE motif are exposed on opposite surfaces of ankyrin repeat seven and could potentially contribute to direct contacts with p300, as suggested by Oswald and colleagues (Oswald et al., 2001). However, Leu 2103 in the LDE motif (Figure 3; L236 in the crystal) is buried within the structure and is the only invariant hydrophobic residue in this helix that participates in the maintenance of the common, elongated hydrophobic core formed by side chains within ankyrin repeat six, the  hairpin linker, and ankyrin repeat seven (Figure 2, mutation 5; Figure 3). Therefore, removal of this hydrophobic side chain by an Ala substitution could decrease considerably the stability of the entire ANK domain (Figures 2 and 3). Compounding the effect of this mutation is the loss of a double salt bridge present between the side chain of Asp in LDE and Arg2085 located in the first ␣ helix in ankyrin repeat seven (R221 in the crystal), which could contribute to the proper folding of this repeat (Figure 2). Destabilization of the ANK domain will most likely affect the direct interaction with MAM; subsequently, if MAM cannot be properly recruited to the NICD/RBPjk complex, the interface required for the recruitment of p300 may not form properly. Support for this comes from analysis of the structural properties of known mutations in the ANK domain (Zweifel et al., 2003; Table 1,
Figure 2). Partial loss-of-function mutations are mapped to surface-exposed residues and thus preserve the overall structure and stability of the ANK domain (all glp-1 mutations in C. elegans, Nsu42c in Drosophila Notch). In contrast, lethal loss-of-function mutations either disrupt tertiary structure (M2) or compromise the stability of the ANK domain (M1). Interestingly, like the LDE mutation, M1 targeted a conserved leucine (L1989A) that makes an analogous contribution to the hydrophobic core near ankyrin repeat four. It is important to mention that residues mutated in M1 and M2 in the fourth ankyrin repeat preserve the integrity of the EP domain. Both mutant NICD proteins bind to RBPjk (Jarriault et al., 1995; and R.K., unpublished data) but are unable to activate transcription (Jarriault et al., 1995; Kato et al., 1997; Kurooka et al., 1998); we interpret that to suggest that the EP domain could not recruit p300 to RBPjk when the ANK domain is destabilized. When MAM is in excess, it can form a trimeric protein complex with RBP and an intact ANK domain missing RAM (Jeffries et al., 2002). Given that addition of an intact ANK domain “rescues” the M1 mutants (Kurooka et al., 1998), the most likely interpretation for this group of experimental observations is that MAM can bridge between the RAM-less NICD and RBPjk, forming an interface for proper binding of p300. If the stability of the ANK domain is compromised by mutations or deletion of
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the seventh repeat that interface fails to form due to global effects on the ANK domain. Finally, several prooncogenic mutations in another ankyrin repeat protein— the tumor suppressor p16INK4A (Tang et al., 1999; Tevelev et al., 1996)—impact amino acids that, like Leu 2103 in Drosophila Notch, are not involved in protein interactions but rather are buried inside the structure and affect the stability of other ankyrin repeats (Tang et al., 2003; Zhang and Peng, 2002). In summary, the demonstration that the ANK domain includes a seventh repeat solidifies a large number of experimental results obtained by various methods into a more coherent view of the relationship between the Notch structure and its function. Previously, the failure to activate transcription by proteins lacking the EP/RE/ AC domain had been attributed to the loss of direct interaction of this domain with HATs or other cofactors; alternatively, in light of the literature reviewed here, it is more likely that these studies mapped the biological importance of the entire ANK domain due to the peculiarities of its folding and the resultant failure to properly recruit MAM (Figure 4). Interestingly, it has been recently noted that phosphorylation near the seventh repeat (Espinosa et al., 2003; Foltz et al., 2002; Ingles-Esteve et al., 2001) may target NICD to degradation and thus reduce its activity; given the newest structural information on what regions constitute the ANK domain, it would be interesting to test if the observed change in NICD half-life is caused by alterations in the seventh ankyrin repeat and thus in overall structural stability of the Notch cleavage product. We move forward through integration of newfound knowledge into our working models. Deletion constructs, similar to the ones highlighted here, are widely used in biological experiments designed to decipher the signaling properties and biological roles of Notch proteins; we need to reexamine those constructs that removed amino acids within the (correctly defined) ANK domain and refine the conclusions drawn from using them. More insight will undoubtedly be gained as additional structural information emerges describing Notch-pathway proteins and their complexes. Finally, a universally applicable lesson is that integration of “hard” structural and biophysical data with the biochemical and genetic observations will be critical for comprehensive understanding of all protein functions.
domains is critical for regulation of HES promoter activity. Mech. Dev. 104, 3–20.
Acknowledgments
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