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

<div><p>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 <i>E</i>.<i>coli</i> cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (R_g) of our NgBR construct to be 18.2 Å with a maximum particle dimension (D_max) of 61.0 Å. <i>Ab initio</i> 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.</p></div

Circular dichroism analysis of monomeric NgBR(79–293) with the mean residue ellipticity (black) fit using the program BESTSEL (red).

<p>The fitting NRMSD (normalized root-mean-square deviation) is 0.016.</p

SAXS analysis of monomeric NgBR(73–293).

<p>(A) In-line SEC-SAXS. The elution profile of NgBR size-exclusion chromatography (top) aligns with the Rg plot of SAXS frames (bottom). (B) Experimental scattering curve of NgBR (blue dots) overlaid with the theoretical scattering curve calculated from UPPs structure (green, χ² = 1.61) and an <i>ab initio</i> dummy atom model (red, χ² = 1.31). (C) Kratky plot of NgBR (blue) overlaid with UPPs theoretical curve. (D) Guinier plot. (E) Pair distance function of NgBR scattering. (F) An <i>ab initio</i> dummy atom model of NgBR (surface) superimposed with UPPs crystal structure (ribbon) (PDB code: 1X08).</p

NgBR shares high sequence similarity with UPPs.

<p>(A) Topological structure of NgBR at the plasma membrane. Domains are colored according to their topological location. Extracellular, red; transmembrane, purple; cytosolic, green. (B) Sequence alignment of NgBR and UPPs. Identical residues are represented as white on black and similar residues are colored in cyan. Residues involved in binding farnesyl pyrophosphate (FPP) are indicated by dots. Secondary structures are numbered based on their sequence position. (C) Ribbon diagram of UPPs structure (PDB code: 1X08). The structure is colored according to the corresponding NgBR domains. Coloring scheme same as 1A. The secondary structural elements are labeled according to 1B. Bound FPP is depicted as sticks. (D) FPP binding pocket in UPPs. (E) A model of UPPs without structural elements corresponding to the extracellular and transmembrane regions of NgBR.</p

A soluble and monodispersed NgBR construct.

<p>(A) NgBR protein constructs. (B) SDS-PAGE analysis of NgBR protein expression and purification. SUMO-NgBR(73–293), left; SUMO-NgBR(137–293), middle; purified NgBR(73–293), right. Lane M, molecular weight marker; U, uninduced cell culture; I, induced cell culture; T, total cell lysate; S, supernatant of cell lysate; 1–3, different monomeric fractions. (C) Elution profile of NgBR(73–293) size-exclusion chromatography.</p