Notch-Jagged signaling complex defined by an interaction mosaic

AbstractThe Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand Jagged1 to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical and structural techniques on the full extracellular complex and targeted sites,we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-13 and Jagged1 C2-EGF3 activation sites, as well as to two additional Jagged1 regions, EGF 8-11 and cysteine-rich domain (CRD). XL-MS and quantitative interaction experiments show that the three Notch1 binding sites on Jagged1 also engage intramolecularly.These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility determined by small-angle X-ray scattering (SAXS), support the formation of backfolded architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal, but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating new routes for therapeutic applications.

Download Full-text

Notch–Jagged signaling complex defined by an interaction mosaic

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2102502118 ◽

2021 ◽

Vol 118 (30) ◽

pp. e2102502118

Author(s):

Matthieu R. Zeronian ◽

Oleg Klykov ◽

Júlia Portell i de Montserrat ◽

Maria J. Konijnenberg ◽

Anamika Gaur ◽

...

Keyword(s):

Developmental Disorders ◽

Regulatory Region ◽

Interaction Network ◽

Activation Mechanism ◽

Signaling Complex ◽

Notch1 Signaling ◽

Rich Domain ◽

X Ray Scattering ◽

Epidermal Growth ◽

Notch1 Receptor

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane–proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8–EGF13 and Jagged1 C2–EGF3 activation sites as well as to two additional Jagged1 regions, EGF8–EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1–Jagged1 activation mechanism and indicating routes for therapeutic applications.

Download Full-text

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

Nature Communications ◽

10.1038/s41467-021-21422-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Timothy H. Tran ◽

Albert H. Chan ◽

Lucy C. Young ◽

Lakshman Bindu ◽

Chris Neale ◽

...

Keyword(s):

Plasma Membrane ◽

Crystal Structures ◽

Regulatory Region ◽

Binding Domain ◽

Published Data ◽

Wild Type ◽

Kras Mutations ◽

Rich Domain ◽

Structural Entity ◽

Cysteine Rich Domain

AbstractThe first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Download Full-text

A Review on Notch Signaling and Colorectal Cancer

Cells ◽

10.3390/cells9061549 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1549

Author(s):

Ashish Tyagi ◽

Arun K. Sharma ◽

Chendil Damodaran

Keyword(s):

Colorectal Cancer ◽

Monoclonal Antibodies ◽

Notch Signaling ◽

Regulatory Region ◽

Severe Toxicity ◽

Oncogenic Signaling ◽

Notch1 Signaling ◽

Notch Receptors ◽

Notch1 Receptor

Colorectal cancer (CRC) has one of the highest mortality rates despite the advancement of treatment options. Aggressive CRC remains difficult to treat owing to the activation of oncogenic signaling pathways such as the Notch signaling pathway. The role of Notch receptors varies according to the difference in their structures; in particular, aberrant activation of Notch1 has been attributed to the severity of CRC. Notch1 activation in CRC is inhibited by small molecule inhibitors that target γ-secretase, an enzyme responsible for the third and last cleavage step of Notch receptors. γ-Secretase also produces the intracellular domain that finally carries out cellular functions by activating downstream effectors. However, most inhibitors block γ-secretase non-selectively and cause severe toxicity. Plant-source-derived small molecules, monoclonal antibodies, biological molecules (such as SiRNAs), and compounds targeting the Notch1 receptor itself or the downstream molecules such as HES1 are some of the options that are in advanced stages of clinical trials. The Negative Regulatory Region (NRR), which plays a central role in the transduction of Notch1 signaling in the event of ligand-dependent and ligand-independent Notch1 processing is also being targeted specifically by monoclonal antibodies (mAbs) to prevent aberrant Notch1 activation. In this review, we discuss the role of Notch1 in CRC, particularly its metastatic phenotype, and how mutations in Notch1, specifically in its NRR region, contribute to the aberrant activation of Notch1 signaling, which, in turn, contributes to CRC pathogenesis. We also discuss prevailing and emerging therapies that target the Notch1 receptor and the NRR region, and we highlight the potential of these therapies in abrogating Notch signaling and, thus, CRC development and progression.

Download Full-text

Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain

Science Advances ◽

10.1126/sciadv.aau4202 ◽

2019 ◽

Vol 5 (7) ◽

pp. eaau4202 ◽

Cited By ~ 4

Author(s):

Janna M. Bigalke ◽

Shintaro Aibara ◽

Robert Roth ◽

Göran Dahl ◽

Euan Gordon ◽

...

Keyword(s):

Calcium Binding ◽

Neurological Diseases ◽

Receptor Activation ◽

Loss Of Function ◽

Calcium Binding Site ◽

Signaling Complex ◽

Rich Domain ◽

Extracellular Region ◽

Domain 3 ◽

Cysteine Rich Domain

Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFRα2 and determined its structure at 5.7-Å resolution by cryo-EM. The proteins form an assembly through RET-GFRα2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFRα2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

Download Full-text

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

10.1101/2020.07.31.231134 ◽

2020 ◽

Cited By ~ 1

Author(s):

Timothy H. Tran ◽

Albert H. Chan ◽

Lucy C. Young ◽

Lakshman Bindu ◽

Chris Neale ◽

...

Keyword(s):

Plasma Membrane ◽

Crystal Structures ◽

Kras Mutation ◽

Regulatory Region ◽

Binding Domain ◽

Published Data ◽

Wild Type ◽

Rich Domain ◽

Structural Entity ◽

Cysteine Rich Domain

ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Download Full-text

Autoregulation of the Raf-1 serine/threonine kinase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9214 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9214-9219 ◽

Cited By ~ 97

Author(s):

Richard E. Cutler ◽

Robert M. Stephens ◽

Misty R. Saracino ◽

Deborah K. Morrison

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Regulatory Region ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rich Domain ◽

Kinase C ◽

Evolutionarily Conserved ◽

Phosphorylation Event ◽

Cysteine Rich Domain

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.

Download Full-text

Cloning of a novel membrane-linked metalloproteinase from human myeloma cells

Biochemical Journal ◽

10.1042/bj3180459 ◽

1996 ◽

Vol 318 (2) ◽

pp. 459-462 ◽

Cited By ~ 14

Author(s):

Norman McKIE ◽

Donna J DALLAS ◽

Tracy EDWARDS ◽

Jane F APPERLEY ◽

Graham G. RUSSELL ◽

...

Keyword(s):

Open Reading Frame ◽

Leader Peptide ◽

Myeloma Cells ◽

Reading Frame ◽

Rich Domain ◽

Epidermal Growth ◽

Transmembrane Sequence ◽

Cysteine Switch ◽

Cysteine Rich Domain ◽

Human Myeloma Cells

We have isolated a novel cDNA from human myeloma cells encoding a member of the reprolysin family of metalloproteinases. Derived amino acid sequence predicts a protein of approx. 76 kDa. The open reading frame predicts the presence of a leader peptide, a pro-peptide with a ‘cysteine switch’, a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich domain, an epidermal growth factor-like domain and a putative transmembrane sequence. Expression of the mRNA for this metalloproteinase has been demonstrated in human myeloma cells.

Download Full-text

Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan

eLife ◽

10.7554/elife.06554 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 49

Author(s):

Tao-Hsin Chang ◽

Fu-Lien Hsieh ◽

Matthias Zebisch ◽

Karl Harlos ◽

Jonathan Elegheert ◽

...

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Wnt Signalling ◽

Rich Domain ◽

X Ray Scattering ◽

Multiple Processes ◽

Density Lipoprotein Receptor ◽

Disease Protein ◽

Cysteine Rich Domain

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8–mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

Download Full-text