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Structural analysis of a type 1 ribosome inactivating protein reveals multiple L‑asparagine‑N‑acetyl‑D‑glucosamine monosaccharide modifications: Implications for cytotoxicity.

Hogg T, Mendel JT, Lavezo JL - Mol Med Rep (2015)

Bottom Line: PAP‑S1aci shares ~95% sequence identity with PAP‑S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP‑S1aci.A rare proline substitution (Pro174) was identified in the active site of PAP‑S1aci, which has no effect on catalytic Glu175 positioning or overall active‑site topology, yet appears to come at the expense of strained main‑chain geometry at the pre‑proline residue Val173.Notably, a rare type of N‑glycosylation was detected consisting of N‑acetyl‑D‑glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP‑S1aci.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Education, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA.

ABSTRACT
Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome‑inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin‑ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X‑ray crystal structure of PAP‑S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP‑S1aci shares ~95% sequence identity with PAP‑S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP‑S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP‑S1aci, which has no effect on catalytic Glu175 positioning or overall active‑site topology, yet appears to come at the expense of strained main‑chain geometry at the pre‑proline residue Val173. Notably, a rare type of N‑glycosylation was detected consisting of N‑acetyl‑D‑glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP‑S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N‑glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA‑binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N‑glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor‑mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

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Active site of PAP-S1aci. (A) Superposition between PAP-S1aci (grey) and PAP-S1 (green; Protein Data Bank code 1gik). Active-site residues for PAP-S1aci are labeled. (B) Electron density features in the vicinity of Val173, a pre-proline Ramachandran outlier according to MolProbity analysis (24). The final model is shown in standard CPK coloring. The final σA-weighted 2Fo-Fc electron density map (cyan; 1.5σ) is overlaid. H-bonds are shown as dashed lines, with corresponding distances between non-hydrogen atoms given in Å.
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f5-mmr-12-04-5737: Active site of PAP-S1aci. (A) Superposition between PAP-S1aci (grey) and PAP-S1 (green; Protein Data Bank code 1gik). Active-site residues for PAP-S1aci are labeled. (B) Electron density features in the vicinity of Val173, a pre-proline Ramachandran outlier according to MolProbity analysis (24). The final model is shown in standard CPK coloring. The final σA-weighted 2Fo-Fc electron density map (cyan; 1.5σ) is overlaid. H-bonds are shown as dashed lines, with corresponding distances between non-hydrogen atoms given in Å.

Mentions: As expected by the high sequence identity between PAP-S1aci and PAP-S1, the overall folds of the two proteins are identical within the limits of experimental detection. An overall Cα least-squares superposition between the two provided a Cα root-mean-square deviation (r.m.s.d.) of 0.48 Å, comparable with the summed cross-validated Luzatti estimates of the coordinate errors in the two structures (0.52 Å). A few small regional Cα displacements on the order of 0.8–1.3 Å can be attributed to localized areas of poor density or differences in crystal packing between the two crystal forms. A Cα geometrical validation of our refined model with MolProbity (24) indicated a significant outlier, Val173 (φ,Ψ= −114°, −83°), which lies outside the small α region of the pre-proline Ramachandran plot. Given that the nearby Glu175 is a conserved active-site residue believed to stabilize the developing oxocarbonium ion during substrate cleavage (15), the present study aimed to determine whether the intervening Pro174 was commonly found in RIP sequences. An extensive protein-protein BLAST search and alignment of non-redundant RIP sequences indicated the residue preceding the catalytic Glu175 was almost invariably Ser or Ala (corresponding to Ser174 in PAP-S1; Fig. 3A). Only three sequences out of all known bacterial and plant RIPs contain a Pro residue in this position: Ribosome inactivating protein 2 from Phytolacca insularis (Korean pokeweed), curcin from Jatropha curcas (spurge family) and ebulin from Sambucus ebulus (Dwarf Elder). Since the geometrically strained Val173 is situated in a break between helices α5 and α6 (Fig. 3A), the present study aimed to determine whether the Pro174 substitution in PAP-S1aci led to any notable structural deviations in the α5/α6 region or if the refined geometry of Val173 is fully supported by the X-ray data. A superposition analysis between the structures of PAP-S1aci and PAP-S1 revealed the Ser→Pro substitution in PAP-S1aci is accommodated without significant alterations in the positioning of Glu175 or in the overall topology of the active site (Fig. 5A). In addition, the lower than average temperature factors in this region (B 2 Val173=15.4 Å ; BProtein=22.3 Å2), coupled with excellent electron density, strongly supports the refined backbone conformation at Val173-Pro174 (Fig. 5B). Although the main-chain conformation does appear to be rather unusual, a more recent statistical analysis of protein pre-Pro geometry indicated that the φ,Ψ values observed for Val173 are within a broad sterically allowed area extending from the α region towards the lower left corner of the Ramachandran plot (36).


Structural analysis of a type 1 ribosome inactivating protein reveals multiple L‑asparagine‑N‑acetyl‑D‑glucosamine monosaccharide modifications: Implications for cytotoxicity.

Hogg T, Mendel JT, Lavezo JL - Mol Med Rep (2015)

Active site of PAP-S1aci. (A) Superposition between PAP-S1aci (grey) and PAP-S1 (green; Protein Data Bank code 1gik). Active-site residues for PAP-S1aci are labeled. (B) Electron density features in the vicinity of Val173, a pre-proline Ramachandran outlier according to MolProbity analysis (24). The final model is shown in standard CPK coloring. The final σA-weighted 2Fo-Fc electron density map (cyan; 1.5σ) is overlaid. H-bonds are shown as dashed lines, with corresponding distances between non-hydrogen atoms given in Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581812&req=5

f5-mmr-12-04-5737: Active site of PAP-S1aci. (A) Superposition between PAP-S1aci (grey) and PAP-S1 (green; Protein Data Bank code 1gik). Active-site residues for PAP-S1aci are labeled. (B) Electron density features in the vicinity of Val173, a pre-proline Ramachandran outlier according to MolProbity analysis (24). The final model is shown in standard CPK coloring. The final σA-weighted 2Fo-Fc electron density map (cyan; 1.5σ) is overlaid. H-bonds are shown as dashed lines, with corresponding distances between non-hydrogen atoms given in Å.
Mentions: As expected by the high sequence identity between PAP-S1aci and PAP-S1, the overall folds of the two proteins are identical within the limits of experimental detection. An overall Cα least-squares superposition between the two provided a Cα root-mean-square deviation (r.m.s.d.) of 0.48 Å, comparable with the summed cross-validated Luzatti estimates of the coordinate errors in the two structures (0.52 Å). A few small regional Cα displacements on the order of 0.8–1.3 Å can be attributed to localized areas of poor density or differences in crystal packing between the two crystal forms. A Cα geometrical validation of our refined model with MolProbity (24) indicated a significant outlier, Val173 (φ,Ψ= −114°, −83°), which lies outside the small α region of the pre-proline Ramachandran plot. Given that the nearby Glu175 is a conserved active-site residue believed to stabilize the developing oxocarbonium ion during substrate cleavage (15), the present study aimed to determine whether the intervening Pro174 was commonly found in RIP sequences. An extensive protein-protein BLAST search and alignment of non-redundant RIP sequences indicated the residue preceding the catalytic Glu175 was almost invariably Ser or Ala (corresponding to Ser174 in PAP-S1; Fig. 3A). Only three sequences out of all known bacterial and plant RIPs contain a Pro residue in this position: Ribosome inactivating protein 2 from Phytolacca insularis (Korean pokeweed), curcin from Jatropha curcas (spurge family) and ebulin from Sambucus ebulus (Dwarf Elder). Since the geometrically strained Val173 is situated in a break between helices α5 and α6 (Fig. 3A), the present study aimed to determine whether the Pro174 substitution in PAP-S1aci led to any notable structural deviations in the α5/α6 region or if the refined geometry of Val173 is fully supported by the X-ray data. A superposition analysis between the structures of PAP-S1aci and PAP-S1 revealed the Ser→Pro substitution in PAP-S1aci is accommodated without significant alterations in the positioning of Glu175 or in the overall topology of the active site (Fig. 5A). In addition, the lower than average temperature factors in this region (B 2 Val173=15.4 Å ; BProtein=22.3 Å2), coupled with excellent electron density, strongly supports the refined backbone conformation at Val173-Pro174 (Fig. 5B). Although the main-chain conformation does appear to be rather unusual, a more recent statistical analysis of protein pre-Pro geometry indicated that the φ,Ψ values observed for Val173 are within a broad sterically allowed area extending from the α region towards the lower left corner of the Ramachandran plot (36).

Bottom Line: PAP‑S1aci shares ~95% sequence identity with PAP‑S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP‑S1aci.A rare proline substitution (Pro174) was identified in the active site of PAP‑S1aci, which has no effect on catalytic Glu175 positioning or overall active‑site topology, yet appears to come at the expense of strained main‑chain geometry at the pre‑proline residue Val173.Notably, a rare type of N‑glycosylation was detected consisting of N‑acetyl‑D‑glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP‑S1aci.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Education, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA.

ABSTRACT
Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome‑inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin‑ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X‑ray crystal structure of PAP‑S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP‑S1aci shares ~95% sequence identity with PAP‑S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP‑S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP‑S1aci, which has no effect on catalytic Glu175 positioning or overall active‑site topology, yet appears to come at the expense of strained main‑chain geometry at the pre‑proline residue Val173. Notably, a rare type of N‑glycosylation was detected consisting of N‑acetyl‑D‑glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP‑S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N‑glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA‑binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N‑glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor‑mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

Show MeSH
Related in: MedlinePlus