Smyd4 Subcellular Localization And Nogo-B Receptor Structure And Function

SMYD proteins are a family of proteins known to possess a methyltransferase activity and has been shown to play important roles in cancer and immunology. Currently there are limited studies about SMYD4 linking it to muscle development and breast cancer. Our lab previously showed that SMYD4 interacts with hsp90 tail. Hsp90 is an important protein in the chaperon complex that is responsible for degrading between 30-40% of proteins through delivering them to the lysosomes. My study aimed to further understand SMYD4 by attempting to proof its’ localization via immunofluorescence microscopy inside the lysosomes of U2OS cells. Our results showed SMYD4 to not be localized inside the lysosomes of U2OS cells. The Nogo-B receptor is known for being essential for angiogenesis where its knockdown results in embryonic lethality due to defects in vascular formation. A recent study also shows NgBR to be involved in the binding and activation of Ras to the plasma membrane. Currently the NgBR crystal structure has not been yet solved but previous studies suggested it to be a type I transmembrane protein. However, BLAST alignment and SAX analysis performed previously by our lab members suggests NgBR to be a well folded cytosolic protein resembling the structure of cis-IPTase UPPS protein. We chose to further confirm our previous data through the purification of NgBR (79-293) and using circular dichroism which indeed showed a high percentage similarity between the secondary structure of NgBR and UPPs. Additionally, we were able to purify a new NgBR (55-293) protein that includes additional amino acids from NgBR extracellular domain. 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. SAX analysis performed using NgBR protein that includes the extracellular and transmembrane domains which 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). Methods such as NMR spectroscopy, electron microscopy, and X-crystallography are currently used to determine protein structure and its interactions with other proteins and ligands. Each of these method bears its’ potential advantages and disadvantages. Although X-ray crystallography has numerous approaches that are used to obtain such as antibody fragments, lipids, carrier proteins, etc. can be used to enhance chances of obtaining a protein crystal the success unpredictability of one single method still hinders protein crystallization attempts. In our study we selected X-ray crystallography as a starting point to solve the 3D structure of Nogo-B receptor ((NgBR) protein. Our study successfully expressed and purified NgBR (79-293). This construct was then used to perform circular dichroism. We further confirmed our previous SAX analysis results that showed NgBR to be well folded in solution and resembles UPPS secondary structure content. These findings are contradictory to the currently acceptable topology of NgBR which classifies it as type I transmembrane protein. To enhance the solubility and expression of NgBR (55-293) we designed and performed small scale expression test on fusion construct MBP-NgBR (55-293). These newly expressed constructs open novel opportunities to obtain crystalize and solve the macromolecular structure of NgBR

Protein crystallization: Eluding the bottleneck of X-ray crystallography

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins

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

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