Rhett A. Kovall

Crystal Structure of the CSL-Notch-Mastermind Ternary Complex Bound to DNA

Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. Here we report the 3.1 A structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator.

CRYSTAL STRUCTURE OF THE NUCLEAR EFFECTOR OF NOTCH SIGNALING, CSL, BOUND TO DNA

Notch signaling is a conserved pathway of communication between neighboring cells that results in cell fate specification, and CSL is the universal transcriptional effector of Notch signaling. The Notch intracellular domain translocates to the nucleus after proteolytic release upon Notch extracellular engagement, and there it displaces corepressors from DNA‐bound CSL and recruits activators of Notch target genes. Here we report the 2.85 Å crystal structure of CSL with a target DNA. CSL comprises three structurally integrated domains: its amino (NTD)‐ and carboxy (CTD)‐terminal domains are strikingly similar to those of Rel transcription factors, but a surprising beta‐trefoil domain (BTD) is inserted between them. CSL‐bound DNA is recognized specifically by conserved residues from NTD and BTD. A hydrophobic pocket on BTD is identified as the likely site of Notch interaction with CSL, which has functional implications for the mechanism of Notch signaling.

Mechanistic insights into Notch receptor signaling from structural and biochemical studies.

Notch proteins are the receptors in a highly conserved signal transduction system used to communicate signals between cells that contact each other. Studies investigating structure-function relationships in Notch signaling have gained substantial momentum in recent years. Here, we summarize the current understanding of the molecular logic of Notch signal transduction, emphasizing structural and biochemical studies of Notch receptors, their ligands, and complexes of intracellular Notch proteins with their target transcription factors. Recent advances in the structure-based modulation of Notch-signaling activity are also discussed.

More complicated than it looks : assembly of Notch pathway transcription complexes

The Notch pathway is a short-range signaling mechanism between neighboring cells that results in changes in gene expression. Extracellular interactions between Notch receptors and ligands trigger proteolytic cleavage of the receptor Notch. Following cleavage, the freed intracellular domain of Notch (NotchIC) moves from the cytoplasm to the nucleus, engaging the DNA-binding transcription factor CBF-1, Su(H), Lag-1 (CSL)—the nuclear effector of the pathway. NotchIC, together with the transcriptional coactivator Mastermind, form a ternary complex with CSL that activates transcription from genes that are responsive to Notch signaling. Illuminating the molecular details that underlie formation of the transcriptionally active CSL–NotchIC–Mastermind ternary complex is key for understanding how genes are turned on in response to a Notch signal. Recently, several studies using biophysical and computational methods have scrutinized how the CSL–NotchIC–Mastermind ternary complex forms and the role individual domains play in this process. These detailed analyses have provided a wealth of molecular insights into the assembly of a Notch pathway active transcription complex but have also raised several intriguing, yet confounding questions. This review will focus on the findings of these recent biophysical studies and provide speculative models that address these unanswered questions.

Type II restriction endonucleases: structural, functional and evolutionary relationships.

Type II restriction endonucleases are a paradigm for site-specific cleavage of DNA. Recent structural analyses, in particular in the presence of various divalent metals, have shed new insight into the mechanisms of catalysis. In addition, during this past year the crystal structure determinations of MutH, lambda-exonuclease and FokI have revealed that these proteins are also members of the same family.

RAM-induced Allostery Facilitates Assembly of a Notch Pathway Active Transcription Complex.

The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

The Notch signaling pathway relies on a proteolytic cascade to release its transcriptionally active intracellular domain, on force to unfold a protective domain and permit proteolysis, on extracellular domain glycosylation to tune the forces exerted by endocytosed ligands, and on a motley crew of nuclear proteins, chromatin modifiers, ubiquitin ligases, and a few kinases to regulate activity and half-life. Herein we provide a review of recent molecular insights into how Notch signals are triggered and how cell shape affects these events, and we use the new insights to illuminate a few perplexing observations.

Notch signaling: genetics and structure

Receptors of the Notch family mediate cell-cell interactions during animal development, and aberrations in Notch signaling have been implicated in human disease. Studies in Caenorhabdits elegans have made essential contributions towards understanding the biological roles and molecular mechanism of this fundamental signaling system. A major development in the field since the original version of this chapter (LIN-12/Notch signaling in C. elegans) has been an explosion in information about the structural biology of Notch signaling; crystallographic determinations of structures, including structures of C. elegans components, have contributed much to the current understanding of molecular mechanism. Thus, here, we not only cover the genetics of Notch in C. elegans, focusing on conserved core components and modulators, we have also included structural information about these components, describing the key events occurring during ligand binding and transcriptional control of target genes. In addition to text, we include Tables listing core components and key modulators of the signaling pathway along with their orthologs in Drosophila and mammals, a Table listing validated target genes in various processes in C. elegans, and animated features to illustrate structural attributes.

Transcriptional Repression in the Notch Pathway: THERMODYNAMIC CHARACTERIZATION OF CSL-MINT (Msx2-INTERACTING NUCLEAR TARGET PROTEIN) COMPLEXES*

The Notch pathway is a conserved cell-to-cell signaling mechanism that mediates cell fate decisions in metazoans. Canonical signaling results in changes in gene expression, which is regulated by the nuclear effector of the pathway CSL (CBF1/RBP-J, Su(H), Lag-1). CSL is a DNA binding protein that functions as either a repressor or an activator of transcription, depending upon whether it is complexed by transcriptional corepressor or coactivator proteins, respectively. In stark contrast to CSL-coactivator complexes, e.g. the transcriptionally active CSL-Notch-Mastermind ternary complex, the structure and function of CSL-corepressor complexes are poorly understood. The corepressor MINT (Msx2-interacting nuclear target protein) has been shown in vivo to antagonize Notch signaling and shown in vitro to biochemically interact with CSL; however, the molecular details of this interaction are only partially defined. Here, we provide a quantitative thermodynamic binding analysis of CSL-MINT complexes. Using isothermal titration calorimetry, we demonstrate that MINT forms a high affinity complex with CSL, and we also delineate the domains of MINT and CSL that are necessary and sufficient for complex formation. Moreover, we show in cultured cells that this region of MINT can inhibit Notch signaling in transcriptional reporter assays. Taken together, our results provide functional insights into how CSL is converted from a repressor to an activator of transcription.

Thermodynamic and structural insights into CSL-DNA complexes

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C‐promoter binding factor 1 (CBF‐1), suppressor of hairless (Su(H)), lin‐12 and glp‐1 (Lag‐1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well‐characterized Notch target gene is hairy and enhancer of split‐1 (HES‐1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head‐to‐head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL‐DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES‐1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES‐1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo.