IADR Abstract Archives
Structural Analysis of a Skp Like Protein (PGN_0301) of Porphyromonas Gingivalis.
Objectives: Porphyromonas gingivalis is one of the key pathogens of periodontal disease. This bacteria secretes gingipains through the secretion systems. Therefore, elucidating the mechanisms of secretion system is crucial for understanding infection and pathogenicity.In previous studies, we identified an Omp-like protein, PGN_0300(SkpA), a chaperone protein important in the secretion mechanism, and found it to be part of an operon from PGN_0296 to PGN_0301 (SkpB). Within this operon, SkpB is also considered an Omp-like protein, similar to PGN_0300 (SkpA), but its function remains unclear. In this research, we investigated the structure and role of SkpB using genetic engineering and X-ray crystallography.
Methods: The recombinant protein of SkpB-His6 was expressed in Escherichia coli and purified using a Ni-NTA column and size-exclusion chromatography. Crystallization of the purified recombinant SkpB was performed and its structure was determined by X-ray diffraction experiments. Mutations were introduced at residues considered important from a structure, and changes in the phenotype were investigated.
Results: The crystal structure of the rSkpB was determined at a 3.3A resolution. SkpB has an α-helical hairpin extension region like a tentacle shape, consisting of α2 and α3 helices, with a characteristic proline residue at the root of the α2 helix (Pro34). Structural comparison predicted that SkpA lacks the loop protrusion present in SkpB, which is important for T9SS in Flavobacterium johnsoniae. SkpB forms a trimer mediated hydrophobic interaction, represented by Tyr135. The inner surface of the SkpB trimer is covered with hydrophobic regions, likely recognizing unfolded proteins. Structural comparisons show that SkpB is similar to the Skp protein of E. coli, and furthermore, the predicted structure of PGN_0299 in the same operon is BamA-like, suggesting that these proteins likely function together. Moreover, the mutated SkpBs were shown to cause a reduction in growth rate and a change in the color of the colonies.
Conclusions: SkpB forms a trimer, indicating a different structure from SkpA. SkpB would play a role in the BamA-like transport system via PGN_0299, based on comparison with E. coli, and this pathway might contribute to the transport of the T9SS protein, as suggested by the color change of the colonies.
Methods: The recombinant protein of SkpB-His6 was expressed in Escherichia coli and purified using a Ni-NTA column and size-exclusion chromatography. Crystallization of the purified recombinant SkpB was performed and its structure was determined by X-ray diffraction experiments. Mutations were introduced at residues considered important from a structure, and changes in the phenotype were investigated.
Results: The crystal structure of the rSkpB was determined at a 3.3A resolution. SkpB has an α-helical hairpin extension region like a tentacle shape, consisting of α2 and α3 helices, with a characteristic proline residue at the root of the α2 helix (Pro34). Structural comparison predicted that SkpA lacks the loop protrusion present in SkpB, which is important for T9SS in Flavobacterium johnsoniae. SkpB forms a trimer mediated hydrophobic interaction, represented by Tyr135. The inner surface of the SkpB trimer is covered with hydrophobic regions, likely recognizing unfolded proteins. Structural comparisons show that SkpB is similar to the Skp protein of E. coli, and furthermore, the predicted structure of PGN_0299 in the same operon is BamA-like, suggesting that these proteins likely function together. Moreover, the mutated SkpBs were shown to cause a reduction in growth rate and a change in the color of the colonies.
Conclusions: SkpB forms a trimer, indicating a different structure from SkpA. SkpB would play a role in the BamA-like transport system via PGN_0299, based on comparison with E. coli, and this pathway might contribute to the transport of the T9SS protein, as suggested by the color change of the colonies.