title: Glutamate provides a key structural contact between reticulon-4 (Nogo-66) and phosphocholine.
abstract: Human reticulon 4 (RTN-4) has been identified as the neurite outgrowth inhibitor (Nogo). This protein contains a span of 66 amino acids (Nogo-66) flanked by two membrane helices at the C-terminus. We previously determined the NMR structure of Nogo-66 in a native-like environment and defined the regions of Nogo-66 expected to be membrane embedded. We hypothesize that aromatic groups and a negative charge hyperconserved among RTNs (Glu26) drive the remarkably strong association of Nogo-66 with a phosphocholine surface. Glu26 is an isolated charge with no counterion provided by nearby protein groups. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. In an aqueous environment, Nogo-66 Glu26Ala is more helical than WT and binds the Nogo receptor with higher affinity. Thus, we can conclude that in the absence of a neutralizing positive charge provided by lipid, the glutamate anion is destabilizing to the Nogo-66 fold. Although the Nogo-66 Glu26Ala free energy of transfer from water into lipid is similar to that of WT, NMR data reveal a dramatic loss of tertiary structure for the mutant in DPC micelles. These data show that Glu26 has a key role in defining the structure of Nogo-66 on a phosphocholine surface. This article is part of a special issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.
authors.affiliation: Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Alhoshani
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Vithayathil
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bandong
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Chrunyk
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Gabriel O
authors.initials: GO
authors.lastname: Moreno
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA; Department of Chemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
authors.affiliation: Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA; Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. Electronic address: mcocco@uci.edu.
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Cocco
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D009185:Myelin Proteins / Q000737:chemistry* / Q000378:metabolism; D000070798:Nogo Proteins; D010455:Peptides / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary
chemical_list: D008565:Membrane Proteins; D008823:Micelles; D009185:Myelin Proteins; D000070798:Nogo Proteins; D010455:Peptides; C000603199:RTN4 protein, human; D010767:Phosphorylcholine; D018698:Glutamic Acid; C028810:dodecylphosphocholine
keywords: Choline; Glutamate; Membrane interface; Nogo-66; Reticulon; Structure