MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors.

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila melanogaster , loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling.

The zinc finger-associated SCAN box is a conserved oligomerization domain.

A number of Cys(2)His(2) zinc finger proteins contain a highly conserved amino-terminal motif termed the SCAN domain. This element is an 80-residue, leucine-rich region that contains three segments strongly predicted to be alpha-helices. In this report, we show that the SCAN motif functions as an oligomerization domain mediating self-association or association with other proteins bearing SCAN domains. These findings suggest that the SCAN domain plays an important role in the assembly and function of this newly defined subclass of transcriptional regulators.

Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers.

A new procedure for the production of a defined library of random mutants is described. Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three 'wrong' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol. Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents. Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent 'marker' base can be included that allows an early estimate of the mutagenesis efficiency. The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing. This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase. Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase. This approach provides a more complete library than a method using chemical mutagenic reagents.

Structural independence of ligand-binding modules five and six of the LDL receptor.

The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.

How can a catalytic lesion be offset? The energetics of two pseudorevertant triosephosphate isomerases.

The reaction energetics of four triosephosphate isomerase mutants are compared with those of the wild-type enzyme. The two primary mutants, E165D and H95N, contain site-specific alterations of active site residues. In one case the active site base has been altered (E165D), and in the other, an active site electrophile has been removed (H95N), yet the major effect in each case is the relative destabilization of the transition states for the two chemical (enolization) steps that constitute the catalytic reaction. When the genes encoding each of these sluggish mutant isomerases were subjected to random mutagenesis using chemical reagents and a selection for isomerases of increased catalytic potency was performed, pseudorevertant enzymes with dramatic increases in activity were found. Remarkably, the same second-site suppressor locus partially corrects each lesion. The E165D,S96P pseudorevertant is a 20-fold better catalyst than the E165D mutant from which it is derived, and the H95N,S96P pseudorevertant is about 60 times more active than its H95N parent. The S96P substitution thus increases the catalytic activity in each of two different contexts, H95N and E165D. The energetic consequences of the S96P change are suprisingly similar in each pseudorevertant. The H95N,S96P enzyme is more effective than H95N at stabilizing the intermediate enediol(ate) phosphate and its flanking transition states. The E165D,S96P enzyme likewise stabilizes the transition states for enolization better than E165D, and this pseudorevertant also forms a tighter enzyme-dihydroxyacetone phosphate complex than its parent. These data show how, in these two cases, the catalytic potency of sluggish mutant enzymes can be improved by second-site changes. The results thus provide the beginnings of a detailed understanding of the kinetic refinement of enzyme catalysts.

Folding determinants of LDL receptor type A modules.

To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5). In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other. LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present. Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered. These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold.

Evidence that familial hypercholesterolemia mutations of the LDL receptor cause limited local misfolding in an LDL-A module pair.

Mutations at conserved sites within the ligand-binding LDL-A modules of the LDL receptor cause the genetic disease familial hypercholesterolemia (FH), and several of these FH mutations in modules five and six prevent the isolated single modules from folding properly to a nativelike three-dimensional structure. Because LDL-A modules occur as a series of contiguous repeats in the LDLR and related proteins, we investigated the impact of two FH mutations in LDL-A module five (D203G and D206E) and two mutations in module six (E219K and D245E) in the context of the covalently connected module five-six pair. HPLC chromatography of the products formed under conditions that efficiently refold the native module five-six pair demonstrate that, for each mutation, a folding defect persists in the module pair. NMR spectroscopy and calcium affinity measurements of the ensemble of misfolded products demonstrate that the unaltered module of each pair can fold to its native structure regardless of the range of misfolded conformations adopted by its mutated neighbor. These findings lend additional support to a model in which individual LDL-A modules of the LDL receptor act as independent structural elements.

Solution structure of the sixth LDL-A module of the LDL receptor.

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of plasma cholesterol into cells and serves as a prototype for an entire class of cell surface receptors. The amino-terminal domain of the receptor consists of seven LDL-A modules; the third through the seventh modules all contribute to the binding of low-density lipoproteins (LDLs). Here, we present the NMR solution structure of the sixth LDL-A module (LR6) from the ligand binding domain of the LDLR. This module, which has little recognizable secondary structure, retains the essential structural features observed in the crystal structure of LDL-A module five (LR5) of the LDLR. Three disulfide bonds, a pair of buried residues forming a hydrophobic "mini-core", and a calcium-binding site that serves to organize the C-terminal lobe of the module all occupy positions in LR6 similar to those observed in LR5. The striking presence of a conserved patch of negative surface electrostatic potential among LDL-A modules of known structure suggests that ligand recognition by these repeats is likely to be mediated in part by electrostatic complementarity of receptor and ligand. Two variants of LR6, identified originally as familial hypercholesterolemia (FH) mutations, have been investigated for their ability to form native disulfide bonds under conditions that permit disulfide exchange. The first, E219K, lies near the amino-terminal end of LR6, whereas the second, D245E, alters one of the aspartate side chains that directly coordinate the bound calcium ion. After equilibration at physiologic calcium concentrations, neither E219K nor D245E folds to a unique disulfide isomer, indicating that FH mutations both within and distant from the calcium-binding site give rise to protein-folding defects.

Stepwise improvements in catalytic effectiveness: independence and interdependence in combinations of point mutations of a sluggish triosephosphate isomerase.

Second-site suppressor changes that improve the catalytic potency of a sluggish mutant of the enzyme triosephosphate isomerase have been examined both individually and in combination. Each of the second-site mutations increases the specific catalytic activity of a triosephosphate isomerase in which the catalytic base, glutamate-165, has been changed to aspartate. These second-site suppressors are G10S, S96P, S96T, E97D, V167D, and G233R. Not one of these changes enhances the value of kcat/Km for the wild-type enzyme, which is consistent with the knowledge that the reaction catalyzed by the wild-type enzyme is already diffusion-controlled. Indeed, two of the changes, S96P and V167D, are catalytically deleterious to the wild-type isomerase. When pairs of second-site suppressors are combined with the primary lesion E165D, six pairs show additive independence while the effects of eight other pairs are less than additive. The sites fall into two clusters: pairs within a cluster always interfere with one another and do not produce additive improvements in catalytic activity, whereas combinations of changes from different clusters tend to be additive in their effects. No combination of second-site suppressor mutations behaves synergistically, though there seems to be no a priori reason to exclude this possibility. Since the catalytic potency of each of the six second-site suppressor mutants can be further improved by the introduction of (at least) one of the other five changes, it is evident that none of the double mutants lies at a local catalytic maximum. In these cases, therefore, the opportunity exists for at least two "steps" of monotonic catalytic improvement along each of six different "paths" in protein space.

Structural features of the low-density lipoprotein receptor facilitating ligand binding and release.

The LDLR (low-density lipoprotein receptor) is a modular protein built from several distinct structural units: LA (LDLR type-A), epidermal growth factor-like and beta-propeller modules. The low pH X-ray structure of the LDLR revealed long-range intramolecular contacts between the propeller domain and the central LA repeats of the ligand-binding domain, suggesting that the receptor changes its overall shape from extended to closed, in response to pH. Here we discuss how the LDLR uses flexibility and rigidity of linkers between modules to facilitate ligand binding and low-pH ligand release.

Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme.

How easy is it to improve the catalytic power of an enzyme? To address this question, the gene encoding a sluggish mutant triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) has been subjected to random mutagenesis over its whole length by using "spiked" oligonucleotide primers. Transformation of an isomerase-minus strain of Escherichia coli was followed by selection of those colonies harboring an enzyme of higher catalytic potency. Six amino acid changes in the Glu-165----Asp mutant of triosephosphate isomerase improve the specific catalytic activity of this enzyme (from 1.3-fold to 19-fold). The suppressor sites are scattered across the sequence (at positions 10, 96, 97, 167, and 233), but each of them is very close to the active site. These experiments show both that there are relatively few single amino acid changes that increase the catalytic potency of this enzyme and that all of these improvements derive from alterations that are in, or very close to, the active site.

A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid.

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.

Two functionally distinct forms of a retroviral receptor explain the nonreciprocal receptor interference among subgroups B, D, and E avian leukosis viruses.

Subgroups B, D, and E avian leukosis viruses (ALV-B, -D, and -E) share the same chicken receptor, TVB(S1), a tumor necrosis factor receptor (TNFR)-related protein. These viruses, however, exhibit nonreciprocal receptor interference (NRI): cells preinfected with ALV-B or ALV-D are resistant to superinfection by viruses of all three subgroups, whereas those pre-infected by ALV-E are resistant only to superinfection by other subgroup E viruses. In this study, we investigated the basis of this phenomenon by characterizing the interaction of TVB(S1) with ALV-B Env or ALV-E Env. Sequential immunoprecipitation analysis using surface envelope immunoglobulin fusion proteins revealed the existence of two separate types of TVB(S1) that are encoded by the same cDNA clone. One form, designated the type 1 receptor, is specific for ALV-B and ALV-E. The other form, the type 2 receptor, is specific for ALV-B. We show that a protein consisting of only the first and second extracellular cysteine-rich domains of TVB(S1) is capable of forming both receptor types. However, the third extracellular cysteine-rich domain is required for efficient formation of the type 1 receptor. We also demonstrate that heterogeneous N-linked glycosylation cannot explain the difference in activities of the two receptor types. The existence of two types of TVB(S1) explains the NRI pattern between ALV-B and -E: subgroup B viruses establish receptor interference with both receptor types, whereas subgroup E viruses interfere only with the type 1 receptor, leaving the type 2 receptor available to mediate subsequent rounds of ALV-B entry. The formation of a TVB receptor type that is specific for cytopathic ALV may also have important implications for understanding how some subgroups of ALV cause cell death.

Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor.

The ligand-binding domain of the LDL receptor consists of seven contiguous LDL-A modules. The fifth of these ligand-binding modules is absolutely required for recognition of both LDL and beta-VLDL particles. A four-residue linker of variable sequence connects each pair of modules, except for modules four and five, which are connected by a 12-residue linker. To provide a more detailed understanding of the structural relationship in a typical pair of functionally important LDL-A repeats of the LDLR, we investigated the backbone dynamics of repeats five (LR5) and six (LR6) alone and in the context of the covalently connected LR5-6 pair. Our results reveal substantial flexibility in the four-residue linker connecting the two repeats in the LR5-6 pair. The intrinsic dynamic behavior of each repeat is essentially unchanged when the repeats are covalently connected. These observations indicate that the relative orientation of repeats in LR5-6 is not fixed. Modeled in an extended conformation, the linker can separate LR5 and LR6 by up to 15 A, a distance that would allow substantial freedom of motion of each repeat with respect to the other in the pair.

A trimeric structural domain of the HIV-1 transmembrane glycoprotein.

Infection with HIV-1 is initiated by fusion of cellular and viral membranes. The gp41 subunit of the HIV-1 envelope plays a major role in this process, but the structure of gp41 is unknown. We have identified a stable, proteinase-resistant structure comprising two peptides, N-51 and C-43, derived from a recombinant protein fragment of the gp41 ectodomain. In isolation, N-51 is predominantly aggregated and C-43 is unfolded. When mixed, however, these peptides associate to form a stable, alpha-helical, discrete trimer of heterodimers. Proteolysis experiments indicate that the relative orientation of the N-51 and C-43 helices in the complex is antiparallel. We propose that N-51 forms an interior, parallel, homotrimeric, coiled-coil core, against which three C-43 helices pack in an antiparallel fashion. We suggest that this alpha-helical, trimeric complex is the core of the fusion-competent state of the HIV-1 envelope.

A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein.

Previous attempts to define the oligomeric state of the HIV and SIV envelope glycoproteins have yielded conflicting results. We have produced in Escherichia coli a recombinant model for the ectodomain of the SIV envelope protein gp41 and have identified a small, trimeric subdomain by proteolytic digestion of this gp41 fragment. The subdomain assembles from two peptide fragments, spanning residues 28-80 (N28-80) and residues 107-149 (C107-149) of SIV gp41. Each of these peptides contains a 4,3-hydrophobic repeat, the hallmark of coiled-coil sequences. Upon mixing, the peptides form a highly helical, trimeric complex [3(N+C)] that resists proteolysis and has a melting temperature (Tm) above 90 degrees C in physiological buffer. The N- and C-terminal fragments are antiparallel to each other in the complex, as judged by the observation that digestion of a variant recombinant protein truncated at the amino terminus yields a C-terminal fragment shortened at its carboxy terminus. The N28-80 peptide contains more positions within the heptad repeat than C107-149 that are predominantly hydrophobic, suggesting that N28-80 is buried in the interior of the complex. We propose that the complex consists of a parallel, trimeric coiled-coil of the N-terminal peptide, encircled by three C-terminal peptide helices arranged in an antiparallel fashion, and that this complex forms a core within the gp41 extracellular domain.

Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits.

Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

The folding and structural integrity of the first LIN-12 module of human Notch1 are calcium-dependent.

Notch1 is a member of a conserved family of large modular type 1 transmembrane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors. The extracellular domain of all Notch receptors have three iterated LIN-12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis. Each LIN-12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues. To begin to understand the structural basis for LIN-12 function, the first LIN-12 module of human Notch1 (rLIN-12.1) has been expressed recombinantly in Escherichia coli and purified in a reduced form. In redox buffers, rLIN-12.1 forms only one disulfide isomer in the presence of millimolar Ca2+ concentrations, whereas multiple disulfide isomers are observed in the presence of Mg2+ and EDTA. Further, mutation of conserved residues N1460, D1475, and D1478 to alanine abolishes Ca2+-dependent folding of this module. Mass spectrometric analysis of partially reduced rLIN-12.1 has been used to deduce that disulfide bonds are formed between the first and fifth (C1449-C1472), second and fourth (C1454-C1467), and third and sixth (C1463-C1479) cysteines of this prototype module. This arrangement is distinct from that observed in other modules, such as EGF and LDL-A, that also contain three disulfide bonds. One-dimensional proton nuclear magnetic resonance shows that Ca2+ induces a dramatic increase in the extent of chemical shift dispersion of the native rLIN-12.1 amide protons, as seen for the Ca2+-binding LDL-A modules. We conclude that Ca2+ is required both for proper folding and for the maintenance of the structural integrity of Notch/LIN-12 modules.