Structures of three ependymin-related proteins suggest their function as a hydrophobic molecule binder

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.

De novo Phasing Xenons Observed in the Frog Ependymin-Related Protein

“…In the human EPDR1 structure, an extended PEG molecule likely from the crystallization condition was observed in this hydrophobic pocket [6]. EPDR1 was also suggested to bind to fatty Crystals 2020, 10, 32 2 of 7 acids presumably via this pocket [7]. Another interesting feature of EPDR1 was a binding site for a metal which was modeled as a bound Ca 2+ [7].…”

“…Recently, two groups including ours have reported the crystal structures of the ependymin-related protein 1 (EPDR1) of human, mouse and frog [6,7]. Ependymin was first discovered in the brain extracellular fluid (ECF) of teleost fish [8], but the orthologous EPDRs are known to exist broadly from protozoa to humans [9].…”

“…While the fish ependymin is secreted into ECF, the EPDRs with a mannose 6-phosphate (M6P) modification in human beings and rodents are found within the lysosome of various tissues [10,11]. The monomeric EPDR1 is made up of two stacking anti-parallel β-sheets with the curvature in one of the layers resulting in creating a deep~6,000 Å 3 hydrophobic pocket [6,7]. This monomeric subunit further associates into a dimer.…”

“…EPDR1 was also suggested to bind to fatty Crystals 2020, 10, 32 2 of 7 acids presumably via this pocket [7]. Another interesting feature of EPDR1 was a binding site for a metal which was modeled as a bound Ca 2+ [7].…”

“…In the previous structural work of frog, mouse and human EPDR1 [7], a single wavelength anomalous diffraction (SAD) was performed on a Xe-pressurized crystal of frog EPDR1 during the phase determination step. Since no analysis of the Xe-bound sites has been performed, we analyze the four bound Xe atoms and their surrounding environment in the frog EPDR1 structure in this study.…”