109 results found
Sammon D, Hohenester E, Leitinger B, 2021, Two-step release of kinase autoinhibition in discoidin domain receptor 1 (vol 117, 22051, 2020), PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 118, ISSN: 0027-8424
McKee KK, Hohenester E, Aleksandrova M, et al., 2021, Organization of the laminin polymer node, Matrix Biology, Vol: 98, Pages: 49-63, ISSN: 0174-173X
Laminin polymerization is a key step of basement membrane assembly that depends on the binding of α, β and γ N-terminal LN domains to form a polymer node. Nodal assembly can be divided into two steps consisting of β- and γ-LN dimerization followed by calcium-dependent addition of the α-LN domain. The assembly and structural organization of laminin-111 LN-LEa segments was examined by size-exclusion chromatography (SEC) and electron microscopy. Triskelion-like structures were observed in negatively-stained images of purified α1/β1/γ1 LN-LEa trimers. Image averaging of these revealed a heel-to-toe organization of the LN domains with angled outward projections of the LEa stem-like domains. A series of single-amino acid substitutions was introduced into the polymerization faces of the α1, β1 and γ1 LN domains followed by SEC analysis to distinguish between loss of β-γ mediated dimerization and loss of α-dependent trimerization (with intact β-γ dimers). Dimer-blocking mutations were confined to the γ1-toe and the β1-heel, whereas the trimer-only-blocking mutations mapped to the γ1-heel, β1-toe and the α1-toe and heel. Thus, in the polymer node the γ1-toe pairs with the β1-heel, the β1-toe pairs with the α1-heel, and the α1-toe pairs with the γ1-heel.
Gao J, Lin P-H, Setare NT, et al., 2021, Exploration of human xylosyltransferase for chemoenzymatic synthesis of proteoglycan linkage region, Organic and Biomolecular Chemistry, Vol: 19, Pages: 3374-3378, ISSN: 1477-0520
Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature, PGs are highly challenging synthetic targets due to the difficulties in integrating the sulfated glycans with the peptide backbone. To expedite the synthesis, herein, the utility of human xylosyltransferase I (XT-I), the enzyme responsible for initiating PG synthesis, has been explored. XT-I was found to be capable of efficiently installing the xylose unit onto a variety of peptide structures on mg scales. Furthermore, an unnatural sugar, i.e., 6-azidoglucose can be transferred by XT-I introducing a reactive handle onto the glycopeptide for selective functionalization. XT-I can be coupled with β-4-galactosyl transferase-7 for one pot synthesis of glycopeptides bearing galactose-xylose disaccharide, paving the way toward efficient chemoenzymatic synthesis of PG glycopeptides and glycoproteins.
Sammon D, Hohenester E, Leitinger B, 2020, Two-step release of kinase autoinhibition in discoidin domain receptor 1, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 22051-22060, ISSN: 0027-8424
Discoidin domain receptor1 (DDR1)is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis.Aberrant DDR1activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity.Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps:rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells.The new insights may be used to develop allosteric, DDR1-specific,kinase inhibitors.
Mittwollen R, Wohlfart S, Park J, et al., 2020, Aberrant splicing as potential modifier of the phenotype of junctional epidermolysis bullosa, JOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VENEREOLOGY, Vol: 34, Pages: 2127-2134, ISSN: 0926-9959
- Author Web Link
- Citations: 4
Hohenester E, 2019, Structural biology of laminins, Essays in Biochemistry, Vol: 63, Pages: 285-295, ISSN: 0071-1365
Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.
Sammon D, Hohenester E, Leitinger B, 2019, The regulation of DDR1 catalysis by its intracellular juxtamembrane region, Spring Meeting of the British-Society-for-Matrix-Biology (BSMB) on Stroma, Niche and Repair, Publisher: WILEY, Pages: A33-A33, ISSN: 0959-9673
Hohenester E, 2019, Laminin G-like domains: dystroglycan-specific lectins, Current Opinion in Structural Biology, Vol: 56, Pages: 56-63, ISSN: 0959-440X
A unique O-mannose-linked glycan on the transmembrane protein dystroglycan binds a number of extracellular matrix proteins containing laminin G-like (LG) domains. The dystroglycan-matrix interaction is essential for muscle function: disrupted biosynthesis of the matrix-binding modification causes several forms of muscular dystrophy. The complete chemical structure of this modification has been deciphered in the past few years. We now know that LG domains bind to a glycosaminoglycan-like polysaccharide of [-3GlcAβ1,3Xylα1-] units, termed matriglycan, that is attached to a highly unusual heptasaccharide linker. X-ray crystallography has revealed the principles of Ca2+-dependent matriglycan binding by LG domains. In this review, the new structural insights are applied to the growing number of LG domain-containing proteins that bind dystroglycan. It is proposed that LG domains be recognised as ‘D-type’ lectins to indicate their conserved function in dystroglycan binding.
Pulido D, Sharma U, Vadon-Le Goff S, et al., 2018, Structural basis for the acceleration of procollagen processing by procollagen C-proteinase enhancer-1, Structure, Vol: 26, Pages: 1384-1392.e3, ISSN: 0969-2126
Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling.
Briggs DC, Hohenester E, 2018, Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1., Structure (London, England : 1993), Vol: 26, Pages: 801-809.e3, ISSN: 0969-2126
Proteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.
- Open Access Link
- Citations: 15
Paracuellos P, Kalamajski S, Bonna A, et al., 2017, Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin, Matrix Biology, Vol: 63, Pages: 106-116, ISSN: 1569-1802
The small leucine-rich proteoglycans (SLRPs) are important regulators of extracellular matrix assembly and cell signalling. We have determined crystal structures at ~2.2 Å resolution of human fibromodulin and chondroadherin, two collagen-binding SLRPs. Their overall fold is similar to that of the prototypical SLRP, decorin, but unlike decorin neither fibromodulin nor chondroadherin forms a stable dimer. A previously identified binding site for integrin α2β1 maps to an α-helix in the C terminal cap region of chondroadherin. Interrogation of the Collagen Toolkits revealed a unique binding site for chondroadherin in collagen II, and no binding to collagen III. A triple-helical peptide containing the sequence GAOGPSGFQGLOGPOGPO (O is hydroxyproline) forms a stable complex with chondroadherin in solution. In fibrillar collagen I and II, this sequence is aligned with the collagen cross-linking site KGHR, suggesting a role for chondroadherin in cross-linking.
Pulido D, Hussain S, Hohenester E, 2017, Crystal structure of the heterotrimeric integrin-binding region of laminin-111, Structure, Vol: 25, Pages: 530-535, ISSN: 1878-4186
Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are importantlaminin receptors, but their binding site on the heterotrimeric lamininsis poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ~50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1chain. The LG domains adopt a triangulararrangement, with the C-terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residuein the γ1 chain tail is surface-exposed and predictedto bind tothe metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrinare likely tobe made by the LG1 and LG2 surfacesadjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans.
Ahmed YA, Yates EA, Moss DJ, et al., 2016, Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity, Molecular Biosystems, Vol: 12, Pages: 3166-3175, ISSN: 1742-206X
Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.
Briggs DC, Yoshida-Moriguchi T, Zheng T, et al., 2016, Structural basis of laminin binding to the LARGE glycans on dystroglycan, Nature Chemical Biology, Vol: 12, Pages: 810-814, ISSN: 1552-4469
Dystroglycan is a highly glycosylated extracellular matrix receptor with essentialfunctions in skeletal muscle and the nervous system. Reduced matrix binding byα-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature ofseveral forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE)synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide ispresent on native α-DG from skeletal muscle. The atomic details of matrix binding wererevealed by a high-resolution crystal structure of laminin G-like (LG) domains 4-5 oflaminin α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygenatoms from both sugars replacing Ca2+-bound water molecules. The chelating bindingmode accounts for the high affinity of this protein-carbohydrate interaction. Theseresults reveal a novel mechanism of carbohydrate recognition and provide a structuralframework for elucidating the mechanisms underlying muscular dystrophy.
Pulido D, Briggs DC, Hua J, et al., 2016, Crystallographic analysis of the laminin β2 short arm reveals how the LF domain is inserted into a regular array of LE domains, Matrix Biology, Vol: 57-58, Pages: 204-212, ISSN: 1569-1802
Laminins are a major constituent of all basement membranes. The polymerisation oflaminins at the cell surface is mediated by the three short arms of the cross-shapedlaminin heterotrimer. The short arms contain repeats of laminin-type epidermalgrowth factor-like (LE) domains, interspersed with globular domains of unknownfunction. A single LF domain is inserted between LE5 and LE6 of the laminin β1 andβ2 chains. We report the crystal structure at 1.85 Å resolution of the laminin β2LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterialfamily 35 carbohydrate binding modules, and more distantly to the L4 domainspresent in the short arms of laminin α and γ chains. An α-helical region mediates theextensive interaction of the LF domain with LE5. The relative arrangement of LE5and LE6 is very similar to that of consecutive LE domains in uninterrupted LEtandems. Fitting atomic models to a low-resolution structure of the first eight domainsof the laminin β1 chain determined by small-angle X-ray scattering suggests adeviation from the regular LE array at the LE4-LE5 junction. These results advanceour understanding of laminin structure.
Hohenester E, Paracuellos P, Briggs DC, et al., 2015, Insights into collagen uptake by C-type mannose receptors from the crystal structure of Endo180 domains 1-4, Structure, Vol: 23, Pages: 2133-2142, ISSN: 1878-4186
The C-type mannose receptor and its homologEndo180 (or uPARAP, for urokinase plasminogenactivator receptor-associated protein) mediate theendocytic uptake of collagen by macrophages and fi-broblasts. This process is required for normal tissueremodeling, but also facilitates the growth anddissemination of tumors. We have determined thecrystal structure at 2.5 A˚ resolution of the N-terminalregion of Endo180, consisting of a ricin-like domain,a fibronectin type II (FN2) domain, and two C-typelectin (CTL) domains. The L-shaped arrangement ofthese domains creates a shallow trench spanningthe FN2 and CTL1 domains, which was shown bymutagenesis to bind triple-helical and denaturedcollagen. Small-angle X-ray scattering showed thatthe L-shaped structure is maintained in solution atneutral and acidic pH, irrespective of calcium ionloading. Collagen binding was equally unaffectedby acidic pH, suggesting that collagen release in endosomesis not regulated by changes within theEndo180 N-terminal region.
Zelina P, Blockus H, Zagar Y, et al., 2014, Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution, Neuron, Vol: 84, Pages: 1258-1272, ISSN: 0896-6273
Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3−/− mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction.
Hohenester E, 2014, Signalling complexes at the cell-matrix interface, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 29, Pages: 10-16, ISSN: 0959-440X
- Author Web Link
- Citations: 13
Xu H, Abe T, Liu JKH, et al., 2014, Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links, Insertions, or Deletions in the Extracellular Juxtamembrane Region, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 13565-13574
- Author Web Link
- Citations: 22
Horejs C-M, Serio A, Purvis A, et al., 2014, Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells, Proceedings of the National Academy of Sciences of the United States of America, Vol: 111, Pages: 5908-5913, ISSN: 0027-8424
The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain–derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs—key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.
Riese SB, Kuehne C, Tedder TF, et al., 2014, Heterotropic Modulation of Selectin Affinity by Allosteric Antibodies Affects Leukocyte Rolling, JOURNAL OF IMMUNOLOGY, Vol: 192, Pages: 1862-1869, ISSN: 0022-1767
- Author Web Link
- Citations: 4
Islam M, Gor J, Perkins SJ, et al., 2013, The Concave Face of Decorin Mediates Reversible Dimerization and Collagen Binding, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 35526-35533
- Author Web Link
- Citations: 27
Yip GMS, Chen Z-W, Edge CJ, et al., 2013, A propofol binding site on mammalian GABA(A) receptors identified by photolabeling (vol 9, pg 715, 2013), NATURE CHEMICAL BIOLOGY, Vol: 9, ISSN: 1552-4450
- Author Web Link
- Citations: 3
Carafoli F, Hohenester E, 2013, Collagen recognition and transmembrane signalling by discoidin domain receptors, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, Vol: 1834, Pages: 2187-2194, ISSN: 1570-9639
- Author Web Link
- Citations: 66
Yip GM, Chen ZW, Edge CJ, et al., 2013, A propofol binding site on mammalian GABAA receptors identified by photolabeling, Nature Chemical Biology, Vol: 9, Pages: 715-720, ISSN: 1552-4469
Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.
Carafoli F, Hamaia SW, Bihan D, et al., 2013, An Activating Mutation Reveals a Second Binding Mode of the Integrin alpha 2 I Domain to the GFOGER Motif in Collagens, PLOS ONE, Vol: 8, ISSN: 1932-6203
- Author Web Link
- Citations: 25
Horejs C, Bertazzo S, Hohenester E, et al., 2013, The cleavage of Laminin-111 by MMP-2 affects early differentiation of murine ESCs and iPS cells, 38th Congress of the Federation-of-European-Biochemical-Societies (FEBS), Publisher: WILEY-BLACKWELL, Pages: 446-446, ISSN: 1742-464X
Hohenester E, Yurchenco PD, 2013, Laminins in basement membrane assembly, CELL ADHESION & MIGRATION, Vol: 7, Pages: 56-63
- Author Web Link
- Citations: 262
Purvis A, Hohenester E, 2012, Laminin Network Formation Studied by Reconstitution of Ternary Nodes in Solution, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 44270-44277
- Author Web Link
- Citations: 26
Manka SW, Carafoli F, Visse R, et al., 2012, Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 12461-12466, ISSN: 0027-8424
- Author Web Link
- Open Access Link
- Citations: 149
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