SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains

• Published in: PloS one Vol. 13; no. 1; p. e0191371
• Main Authors: Holcomb, Joshua, Doughan, Maysaa, Spellmon, Nicholas, Lewis, Brianne, Perry, Emerson, Zhang, Yingxue, Nico, Lindsey, Wan, Junmei, Chakravarthy, Srinivas, Shang, Weifeng, Miao, Qing, Stemmler, Timothy, Yang, Zhe
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18-01-2018; Public Library of Science (PLoS)
• Subjects: Angiogenesis; BASIC BIOLOGICAL SCIENCES; Binding; Biochemistry; Biology and Life Sciences; Cell growth; cell membranes; Cell receptors; Cellular signal transduction; Cholesterol; Circular dichroism; circular dichroism spectroscopy; Construction; crystal structure; Data analysis; Dichroism; Distribution functions; E coli; endoplasmic reticulum; Genetic aspects; Health aspects; Homeostasis; Lipids; Liver cancer; Localization; Medicine; Membrane proteins; Molecular biology; Molecular weight; Molecular weight distribution; Nogo protein; Pharmaceutical sciences; Physical Sciences; Plasma; Protein binding; Protein structure; Proteins; Ras protein; recombinant proteins; Research and Analysis Methods; Secondary structure; Signaling; size-exclusion chromatography; Small angle X ray scattering; small-angle scattering; Structural members; Topology; Transmembrane domains; Wound healing; X-ray scattering
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0191371

The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax) of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD) analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.