Structure
Previews Refining a Jagged Edge Stephen C. Blacklow1,2,3,* 1Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston, MA 02115, USA of Cancer Biology, Dana Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA 3Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.11.001 2Department
In a recent Cell Reports article, Chillakuri and colleagues showed that the N-terminal ‘‘MNNL’’ domain of the essential Notch ligand Jagged1 resembles a C2 domain and relies on calcium binding to facilitate productive signal transduction. Notch signaling is a conserved cell-cell EGF repeats 1 and 2, are required for the MNNL domain plays an important communication system that plays essen- binding to Notch receptors and produc- role in ligand-dependent Notch signaling, tial roles in development and tissue tive signaling (Andrawes et al., 2013; however, how it participates in Notch homeostasis in all metazoan organisms. Parks et al., 2006; Shimizu et al., 1999). activation has remained elusive. In addition, dysregulated Notch signaling Similarly, loss-of-function missense In a recent Cell Reports article, Chillahas been implicated in the pathogenesis mutations of Jagged1, which result in kuri et al. (2013) address the structure of a number of human diseases, including the developmental disorder Alagille’s and function of the MNNL domain of cancer, underscoring its critical impor- syndrome, occur most frequently in the Jagged1 through a combination of tance in human biology (Aster et al., MNNL domain, the DSL domain, and the structural, biochemical, and cell-based 2008). Notch signals are initiated when first two EGF repeats (Chillakuri et al., assays. First, they determined the ligands of the Delta or Jagged/Serrate 2012). Despite accruing evidence that X-ray crystal structure of a Jagged1 families on a signal-sending fragment that encompasses cell bind to Notch receptors the MNNL, DSL, and first on signal-receiving cells. three EGF-like modules (here Ligand engagement triggers denoted JAG1N-EGF3). The structure bears some resemregulated proteolysis of blance to the Olympic torch, Notch receptors, leading to with the MNNL domain as release of the Notch intrathe flame and the DSL-EGF3 cellular domain from the region (solved previously; membrane and its eventual Cordle et al., 2008) acting as entry into the nucleus, where the torch handle (Figure 1A). it regulates transcription of The key new structural Notch target genes (see, for finding, entirely unexpected example, Bray, 2006 and based on sequence alignKopan and Ilagan, 2009 for ment or use of other inforcomprehensive reviews). matics tools, is that the Mammals have three DeltaMNNL domain bears striking like ligands and two Jagged structural similarity to C2 ligands, both homologous to domains. Canonical C2 doDrosophila Serrate. These limains, such as those found gands (collectively referred in protein kinase C (PKCa: to as ‘‘DSL’’ ligands) have a Protein data Bank [PDB] ID shared modular organization, code 1DSY) or Munc13 which includes an N-terminal Figure 1. Structure of the Jagged N-terminal Region and (PDB ID code 3KWU) are b MNNL (module at the N-terComparison with C2 Domains from PKCa and MUNC13 sandwich structures that typiminus of Notch ligands) (A) Transparent surface over a ribbon representation of the JAG1N-EGF3 fragcally contain one or two caldomain, a cysteine-rich DSL ment. The MNNL domain is orange, and the DSL-EGF3 stem region is colored blue. The bound calcium ion is gray. The key site of interaction with Notch cium binding sites derived (Delta, Serrate, Lag-2) modreceptors is indicated at the right. from either the b2-b3 and ule, and 6–16 EGF-like re(B) Structural comparison of the Jagged1 MNNL domain (top, gold) with the C2 b6-b7 loops (PKCa) or the peats. Previous studies have domains of Munc13 (middle, cyan) and PKCa (bottom, green). The view shown is rotated clockwise by approximately 90 from that in (A). The backbone is b1-b2 and b5-b6 loops established that the MNNL rendered with a ribbon diagram; side chains participating in calcium coordina(Munc13) at one end of and DSL elements of both tion are illustrated as sticks; and calcium ions are rendered as gray spheres. the domain (Figure 1B). Most Drosophila and mammalian liThe carbon backbone of the phosphatidylserine bound to the PKCa C2 domain is yellow, and heteroatoms are in CPK colors. C2 domains are intracellular, gands, along with at least 2100 Structure 21, December 3, 2013 ª2013 Elsevier Ltd All rights reserved
Structure
Previews and the calcium-binding loops in these proteins often act as switches for recruiting the C2 domains to the plasma membrane by creating binding sites for phospholipids, such as phosphatidylserine, upon calcium loading (Lemmon, 2008). What about the calcium-binding properties of the Jagged1 MNNL domain, and the implications of calcium loading for ligand-induced Notch signaling? A second X-ray crystal structure of the JAG1N-EGF3 fragment solved in the presence of calcium reveals a bound Ca2+ ion in a binding site formed by acidic residues derived from the b1-b2 and b5-b6 loops, as predicted based on alignment with Munc13. The authors then proceed to show that JAG1N-EGF3, as well as homologous regions of human Delta-like 1 and Drosophila Serrate, bind liposomes in a calcium-dependent fashion and that deletion of the MNNL domain reduces liposome binding to background levels. Moreover, mutations that alter the calcium binding residues do not appear to affect the affinity of these proteins for Notch receptors, but the mutated proteins are nevertheless deficient at inducing Notch signal trans-
duction in a reporter-based assay for activity. Together, these findings appear to support the proposal that calcium loading of the C2 domain facilitates a lipid-binding step that is important for Notch ligand function, but several questions still remain. Does the MNNL domain of Jagged1 directly engage the membrane of the signal-receiving cell that expresses the Notch receptor? Or, alternatively, could phospholipid binding by Jagged1 be a proxy interaction for the true calcium-dependent partner of the MNNL domain, perhaps another protein or even a complementary trigger site on Notch itself? The Cell Reports article by Chillakuri et al. (2013) not only gives new insights into the structure and biochemical properties of Notch ligands, but also opens the door to new lines of experimentation in the Notch field that should provide definitive answers to these and other questions in the near future.
Aster, J.C., Pear, W.S., and Blacklow, S.C. (2008). Annu. Rev. Pathol. 3, 587–613. Bray, S.J. (2006). Nat. Rev. Mol. Cell Biol. 7, 678–689. Chillakuri, C.R., Sheppard, D., Lea, S.M., and Handford, P.A. (2012). Semin. Cell Dev. Biol. 23, 421–428. Chillakuri, C.R., Sheppard, D., Ilagen, M.X.G., Holt, L., Abbott, F., Liang, S., Kopan, R., Handford, P.A., and Lea, S.M. (2013). Cell Reports. Published online November 14, 2013. http://dx.doi.org/ 10.1016/j.celrep.2013.10.029. Cordle, J., Johnson, S., Tay, J.Z.Y., Roversi, P., Wilkin, M.B., de Madrid, B.H., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., et al. (2008). Nat. Struct. Mol. Biol. 15, 849–857. Kopan, R., and Ilagan, M.X.G. (2009). Cell 137, 216–233. Lemmon, M.A. (2008). Nat. Rev. Mol. Cell Biol. 9, 99–111. Parks, A.L., Stout, J.R., Shepard, S.B., Klueg, K.M., Dos Santos, A.A., Parody, T.R., Vaskova, M., and Muskavitch, M.A. (2006). Genetics 174, 1947–1961.
REFERENCES Andrawes, M.B., Xu, X., Liu, H., Ficarro, S.B., Marto, J.A., Aster, J.C., and Blacklow, S.C. (2013). J Biol Chem. 288, 25477–25489.
Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y., and Hirai, H. (1999). J. Biol. Chem. 274, 32961– 32969.
Structure 21, December 3, 2013 ª2013 Elsevier Ltd All rights reserved 2101
Previews Refining a Jagged Edge Stephen C. Blacklow1,2,3,* 1Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston, MA 02115, USA of Cancer Biology, Dana Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA 3Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.11.001 2Department
In a recent Cell Reports article, Chillakuri and colleagues showed that the N-terminal ‘‘MNNL’’ domain of the essential Notch ligand Jagged1 resembles a C2 domain and relies on calcium binding to facilitate productive signal transduction. Notch signaling is a conserved cell-cell EGF repeats 1 and 2, are required for the MNNL domain plays an important communication system that plays essen- binding to Notch receptors and produc- role in ligand-dependent Notch signaling, tial roles in development and tissue tive signaling (Andrawes et al., 2013; however, how it participates in Notch homeostasis in all metazoan organisms. Parks et al., 2006; Shimizu et al., 1999). activation has remained elusive. In addition, dysregulated Notch signaling Similarly, loss-of-function missense In a recent Cell Reports article, Chillahas been implicated in the pathogenesis mutations of Jagged1, which result in kuri et al. (2013) address the structure of a number of human diseases, including the developmental disorder Alagille’s and function of the MNNL domain of cancer, underscoring its critical impor- syndrome, occur most frequently in the Jagged1 through a combination of tance in human biology (Aster et al., MNNL domain, the DSL domain, and the structural, biochemical, and cell-based 2008). Notch signals are initiated when first two EGF repeats (Chillakuri et al., assays. First, they determined the ligands of the Delta or Jagged/Serrate 2012). Despite accruing evidence that X-ray crystal structure of a Jagged1 families on a signal-sending fragment that encompasses cell bind to Notch receptors the MNNL, DSL, and first on signal-receiving cells. three EGF-like modules (here Ligand engagement triggers denoted JAG1N-EGF3). The structure bears some resemregulated proteolysis of blance to the Olympic torch, Notch receptors, leading to with the MNNL domain as release of the Notch intrathe flame and the DSL-EGF3 cellular domain from the region (solved previously; membrane and its eventual Cordle et al., 2008) acting as entry into the nucleus, where the torch handle (Figure 1A). it regulates transcription of The key new structural Notch target genes (see, for finding, entirely unexpected example, Bray, 2006 and based on sequence alignKopan and Ilagan, 2009 for ment or use of other inforcomprehensive reviews). matics tools, is that the Mammals have three DeltaMNNL domain bears striking like ligands and two Jagged structural similarity to C2 ligands, both homologous to domains. Canonical C2 doDrosophila Serrate. These limains, such as those found gands (collectively referred in protein kinase C (PKCa: to as ‘‘DSL’’ ligands) have a Protein data Bank [PDB] ID shared modular organization, code 1DSY) or Munc13 which includes an N-terminal Figure 1. Structure of the Jagged N-terminal Region and (PDB ID code 3KWU) are b MNNL (module at the N-terComparison with C2 Domains from PKCa and MUNC13 sandwich structures that typiminus of Notch ligands) (A) Transparent surface over a ribbon representation of the JAG1N-EGF3 fragcally contain one or two caldomain, a cysteine-rich DSL ment. The MNNL domain is orange, and the DSL-EGF3 stem region is colored blue. The bound calcium ion is gray. The key site of interaction with Notch cium binding sites derived (Delta, Serrate, Lag-2) modreceptors is indicated at the right. from either the b2-b3 and ule, and 6–16 EGF-like re(B) Structural comparison of the Jagged1 MNNL domain (top, gold) with the C2 b6-b7 loops (PKCa) or the peats. Previous studies have domains of Munc13 (middle, cyan) and PKCa (bottom, green). The view shown is rotated clockwise by approximately 90 from that in (A). The backbone is b1-b2 and b5-b6 loops established that the MNNL rendered with a ribbon diagram; side chains participating in calcium coordina(Munc13) at one end of and DSL elements of both tion are illustrated as sticks; and calcium ions are rendered as gray spheres. the domain (Figure 1B). Most Drosophila and mammalian liThe carbon backbone of the phosphatidylserine bound to the PKCa C2 domain is yellow, and heteroatoms are in CPK colors. C2 domains are intracellular, gands, along with at least 2100 Structure 21, December 3, 2013 ª2013 Elsevier Ltd All rights reserved
Structure
Previews and the calcium-binding loops in these proteins often act as switches for recruiting the C2 domains to the plasma membrane by creating binding sites for phospholipids, such as phosphatidylserine, upon calcium loading (Lemmon, 2008). What about the calcium-binding properties of the Jagged1 MNNL domain, and the implications of calcium loading for ligand-induced Notch signaling? A second X-ray crystal structure of the JAG1N-EGF3 fragment solved in the presence of calcium reveals a bound Ca2+ ion in a binding site formed by acidic residues derived from the b1-b2 and b5-b6 loops, as predicted based on alignment with Munc13. The authors then proceed to show that JAG1N-EGF3, as well as homologous regions of human Delta-like 1 and Drosophila Serrate, bind liposomes in a calcium-dependent fashion and that deletion of the MNNL domain reduces liposome binding to background levels. Moreover, mutations that alter the calcium binding residues do not appear to affect the affinity of these proteins for Notch receptors, but the mutated proteins are nevertheless deficient at inducing Notch signal trans-
duction in a reporter-based assay for activity. Together, these findings appear to support the proposal that calcium loading of the C2 domain facilitates a lipid-binding step that is important for Notch ligand function, but several questions still remain. Does the MNNL domain of Jagged1 directly engage the membrane of the signal-receiving cell that expresses the Notch receptor? Or, alternatively, could phospholipid binding by Jagged1 be a proxy interaction for the true calcium-dependent partner of the MNNL domain, perhaps another protein or even a complementary trigger site on Notch itself? The Cell Reports article by Chillakuri et al. (2013) not only gives new insights into the structure and biochemical properties of Notch ligands, but also opens the door to new lines of experimentation in the Notch field that should provide definitive answers to these and other questions in the near future.
Aster, J.C., Pear, W.S., and Blacklow, S.C. (2008). Annu. Rev. Pathol. 3, 587–613. Bray, S.J. (2006). Nat. Rev. Mol. Cell Biol. 7, 678–689. Chillakuri, C.R., Sheppard, D., Lea, S.M., and Handford, P.A. (2012). Semin. Cell Dev. Biol. 23, 421–428. Chillakuri, C.R., Sheppard, D., Ilagen, M.X.G., Holt, L., Abbott, F., Liang, S., Kopan, R., Handford, P.A., and Lea, S.M. (2013). Cell Reports. Published online November 14, 2013. http://dx.doi.org/ 10.1016/j.celrep.2013.10.029. Cordle, J., Johnson, S., Tay, J.Z.Y., Roversi, P., Wilkin, M.B., de Madrid, B.H., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., et al. (2008). Nat. Struct. Mol. Biol. 15, 849–857. Kopan, R., and Ilagan, M.X.G. (2009). Cell 137, 216–233. Lemmon, M.A. (2008). Nat. Rev. Mol. Cell Biol. 9, 99–111. Parks, A.L., Stout, J.R., Shepard, S.B., Klueg, K.M., Dos Santos, A.A., Parody, T.R., Vaskova, M., and Muskavitch, M.A. (2006). Genetics 174, 1947–1961.
REFERENCES Andrawes, M.B., Xu, X., Liu, H., Ficarro, S.B., Marto, J.A., Aster, J.C., and Blacklow, S.C. (2013). J Biol Chem. 288, 25477–25489.
Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y., and Hirai, H. (1999). J. Biol. Chem. 274, 32961– 32969.
Structure 21, December 3, 2013 ª2013 Elsevier Ltd All rights reserved 2101
