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IUCrJ
2019 Jun 20;6Pt 4:729-739. doi: 10.1107/S2052252519007668.
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Structures of three ependymin-related proteins suggest their function as a hydrophobic molecule binder.
Park JK
,
Kim KY
,
Sim YW
,
Kim YI
,
Kim JK
,
Lee C
,
Han J
,
Kim CU
,
Lee JE
,
Park S
.
Abstract
Ependymin was first discovered as a predominant protein in brain extracellular fluid in fish and was suggested to be involved in functions mostly related to learning and memory. Orthologous proteins to ependymin called ependymin-related proteins (EPDRs) have been found to exist in various tissues from sea urchins to humans, yet their functional role remains to be revealed. In this study, the structures of EPDR1 from frog, mouse and human were determined and analyzed. All of the EPDR1s fold into a dimer using a monomeric subunit that is mostly made up of two stacking antiparallel β-sheets with a curvature on one side, resulting in the formation of a deep hydrophobic pocket. All six of the cysteine residues in the monomeric subunit participate in the formation of three intramolecular disulfide bonds. Other interesting features of EPDR1 include two asparagine residues with glycosylation and a Ca2+-binding site. The EPDR1 fold is very similar to the folds of bacterial VioE and LolA/LolB, which also use a similar hydrophobic pocket for their respective functions as a hydrophobic substrate-binding enzyme and a lipoprotein carrier, respectively. A further fatty-acid binding assay using EPDR1 suggests that it indeed binds to fatty acids, presumably via this pocket. Additional interactome analysis of EPDR1 showed that EPDR1 interacts with insulin-like growth factor 2 receptor and flotillin proteins, which are known to be involved in protein and vesicle translocation.
Figure 1. The overall structures of human, mouse and frog EPDR1. A ribbon diagram of human EPDR1 is shown (in yellow) with secondary-structure elements indicated. The locations of cysteines (C1–C6) and the disulfide bonds are also shown. A ribbon diagram of mouse EPDR1 is shown (in green) with two asparagine residues and their NAG glycosylation shown as stick models. Although other types of glycosylation were observed at Asn182, only NAG is shown for clarity (see Fig. 3 ▸ for more on glycosylation). A ribbon diagram of frog EPDR1 is shown (in blue) with Ca2+ ions (shown as spheres), four Ca2+-binding waters (shown as spheres) and direct Ca2+-interacting residues (shown in stick representation). The details of the interaction network stabilizing the bound Ca2+ ions in frog EPDR1 are shown in Fig. 3 ▸(c). Note that the same colors will be used throughout the figures. The superposed structures of the three EPDR1s are also shown. The superimposed EPDR1 structures show Cα r.m.s.d.s of 0.8 Å (human versus mouse), 1.1 Å (human versus frog) and 1.1 Å (mouse versus frog).
Figure 2. The overall dimeric structure of human EPDR1. Ribbon diagrams of human EPDR1 as dimers are shown as three different views. Secondary-structure elements are labeled and the locations of cysteine (C1–C6)-mediated disulfide bonds are also shown.
Figure 3. Views of the asparagine residues with glycosylations (in mouse EPDR1) (a, b) and the Ca2+-binding site (in frog EPDR1) (c) with experimental electron densities. (a) In mouse EPDR1, Asn130 is clearly seen to have a NAG modification. A stimulated-annealing OMIT map of F
o − F
c difference density was contoured at 1.1σ. (b) Also in mouse EPDR1, Asn182 is clearly seen to have a NAG–FUC–NAG modification. A stimulated-annealing OMIT map of F
o − F
c difference density was contoured at 1.1σ. (c) In frog EPDR1, Ca2+ was found to be octahedrally coordinated by four water molecules and other nearby atoms of Asp121 and Pro122. The water molecules coordinated to Ca2+ are further stabilized by tight hydrogen-bonding interactions with the nearby residues Asp124, Glu175 and Tyr177. A stimulated-annealing OMIT map of F
o − F
c difference density was contoured at 3.0σ.
Figure 4. Surface colored by atom type (a) and charge-smoothened vacuum contact electrostatic surface (b) of human EPDR1. (a) The surface rendering (N atoms in blue, O atoms in red and C atoms in yellow) of dimeric human EPDR1 shows a deep hydrophobic cleft. The approximate regions of the hydrophobic pockets are circled in white. (b) The surface of negative and positive electrostatic potential patches generated using a charge-smoothened contact potential in PyMOL more clearly illustrates the hydrophobic pocket located within each of the monomeric subunits in the EPDR1 dimer. [Note that the negative (red) and positive (blue) charges scaled in K
B
T/ec units at pH 7 (K
B, Boltzmann constant; ec, charge on the electron) are only qualitatively useful]. The boundaries of the hydrophobic pocket entrances are indicated in black.
Figure 5. Fatty-acid binding measured by the displacement of a fluorescent probe (1,8-ANS) bound to human EPDR1. Fatty-acid binding to human EPDR1 was inferred by measuring the three fatty-acid (C6, caproic acid; C12, lauric acid; C18, stearic acid) concentrations necessary to replace 50% of 1,8-ANS (IC50). Note that the C6 and C12 displacement studies were performed in 0.5% ethanol buffer and that the C18 displacement study was performed in 2% ethanol buffer owing to the limited solubility of C18 in water.
Adams,
A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen-activated protein kinase pathway to activate c-Jun N-terminal kinase and a functional AP-1 containing c-Jun and c-Fos proteins in mouse NB2a cells.
2003, Pubmed
Adams,
A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen-activated protein kinase pathway to activate c-Jun N-terminal kinase and a functional AP-1 containing c-Jun and c-Fos proteins in mouse NB2a cells.
2003,
Pubmed
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010,
Pubmed
Aït-Slimane,
Basolateral internalization of GPI-anchored proteins occurs via a clathrin-independent flotillin-dependent pathway in polarized hepatic cells.
2009,
Pubmed
Babuke,
Hetero-oligomerization of reggie-1/flotillin-2 and reggie-2/flotillin-1 is required for their endocytosis.
2009,
Pubmed
Benowitz,
Localization of a brain protein metabolically linked with behavioral plasticity in the goldfish.
1977,
Pubmed
Brown,
Interactions of IGF-II with the IGF2R/cation-independent mannose-6-phosphate receptor mechanism and biological outcomes.
2009,
Pubmed
Cox,
MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
2008,
Pubmed
Cox,
Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.
2014,
Pubmed
Cremona,
Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.
2011,
Pubmed
Della Valle,
Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method.
2006,
Pubmed
Emsley,
Coot: model-building tools for molecular graphics.
2004,
Pubmed
Ganss,
Calcium-induced conformational transition of trout ependymins monitored by tryptophan fluorescence.
2009,
Pubmed
Ganss,
Calcium binding to sialic acids and its effect on the conformation of ependymins.
1993,
Pubmed
Hirano,
Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein.
2008,
Pubmed
Hoffmann,
Ependymins: meningeal-derived extracellular matrix proteins at the blood-brain barrier.
1996,
Pubmed
Holm,
Dali: a network tool for protein structure comparison.
1995,
Pubmed
In,
RNF20/40-mediated eEF1BδL monoubiquitylation stimulates transcription of heat shock-responsive genes.
2019,
Pubmed
Kane,
A simple assay for intracellular lipid-binding proteins using displacement of 1-anilinonaphthalene 8-sulfonic acid.
1996,
Pubmed
Kim,
High-pressure cooling of protein crystals without cryoprotectants.
2005,
Pubmed
Kollmann,
Identification of novel lysosomal matrix proteins by proteome analysis.
2005,
Pubmed
Krissinel,
Inference of macromolecular assemblies from crystalline state.
2007,
Pubmed
Königstorfer,
Biosynthesis of ependymins from goldfish brain.
1989,
Pubmed
Langer,
Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.
2008,
Pubmed
Langhorst,
Scaffolding microdomains and beyond: the function of reggie/flotillin proteins.
2005,
Pubmed
Laskowski,
PDBsum: a Web-based database of summaries and analyses of all PDB structures.
1997,
Pubmed
Lübke,
Proteomics of the lysosome.
2009,
Pubmed
McCoy,
Phaser crystallographic software.
2007,
Pubmed
Morrow,
Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation.
2002,
Pubmed
Murshudov,
REFMAC5 for the refinement of macromolecular crystal structures.
2011,
Pubmed
Müller-Schmid,
Ependymins from the cerebrospinal fluid of salmonid fish: gene structure and molecular characterization.
1992,
Pubmed
Neumann-Giesen,
Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression.
2004,
Pubmed
Nimmrich,
The novel ependymin related gene UCC1 is highly expressed in colorectal tumor cells.
2001,
Pubmed
Otwinowski,
Processing of X-ray diffraction data collected in oscillation mode.
1997,
Pubmed
Rappsilber,
Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
2007,
Pubmed
Riento,
Endocytosis of flotillin-1 and flotillin-2 is regulated by Fyn kinase.
2009,
Pubmed
Ryan,
The violacein biosynthetic enzyme VioE shares a fold with lipoprotein transporter proteins.
2008,
Pubmed
Schmidt,
Cell-adhesion molecules in memory formation.
1995,
Pubmed
Shashoua,
CMX-8933, a peptide fragment of the glycoprotein ependymin, promotes activation of AP-1 transcription factor in mouse neuroblastoma and rat cortical cell cultures.
2001,
Pubmed
Shashoua,
Ependymin, a brain extracellular glycoprotein, and CNS plasticity.
1991,
Pubmed
Shashoua,
Brain protein metabolism and the acquisition of new patterns of behavior.
1977,
Pubmed
Shashoua,
Brain metabolism and the acquisition of new behaviors. I. Evidence for specific changes in the pattern of protein synthesis.
1976,
Pubmed
Shashoua,
Identification of specific changes in the pattern of brain protein synthesis after training.
1976,
Pubmed
Shimamoto,
Functional characterization of FABP3, 5 and 7 gene variants identified in schizophrenia and autism spectrum disorder and mouse behavioral studies.
2014,
Pubmed
Sleat,
The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.
2005,
Pubmed
Sleat,
Identification of sites of mannose 6-phosphorylation on lysosomal proteins.
2006,
Pubmed
Sleat,
Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.
1996,
Pubmed
Sneddon,
Molecular correlates of social dominance: a novel role for ependymin in aggression.
2011,
Pubmed
Stivala,
Automatic generation of protein structure cartoons with Pro-origami.
2011,
Pubmed
Stuermer,
Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2.
2001,
Pubmed
Suárez-Castillo,
Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.
2004,
Pubmed
,
Echinobase
Suárez-Castillo,
Molecular evolution of the ependymin protein family: a necessary update.
2007,
Pubmed
Takeda,
Crystal structures of bacterial lipoprotein localization factors, LolA and LolB.
2003,
Pubmed
Tang,
Cold-induced ependymin expression in zebrafish and carp brain: implications for cold acclimation.
1999,
Pubmed
Tribl,
"Subcellular proteomics" of neuromelanin granules isolated from the human brain.
2005,
Pubmed
Tyanova,
The Perseus computational platform for comprehensive analysis of (prote)omics data.
2016,
Pubmed
Uhlen,
Towards a knowledge-based Human Protein Atlas.
2010,
Pubmed
Wei,
Crystal structures of human lysosomal EPDR1 reveal homology with the superfamily of bacterial lipoprotein transporters.
2019,
Pubmed
