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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
.
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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.
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