Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1-70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147-312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1-70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1-70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.